Structural basis for tRNA-dependent sterol aminoacylation underlying cell membrane integrity

Structural basis for tRNA-dependent sterol aminoacylation underlying cell membrane integrity | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Structural basis for tRNA-dependent sterol aminoacylation underlying cell membrane integrity Osamu Nureki, Hanako Murayama, Michihiro Nishimura, Yoshiaki Kise, and 12 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7291846/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract Ergosteryl-3β-O-L-aspartate synthase (ErdS) catalyzes tRNA-dependent aspartylation of ergosterol, a lipid essential for fungal cell membrane integrity. However, the functional significance of ergosteryl-aspartate and the molecular mechanisms underlying its synthesis remain unclear. Here, we show that ErdS localization is highly dynamic and provide evidence that Erg-Asp is required for proper hyphal growth, sporulation, and spore germination, and likely influences stress tolerance. The cryo-electron microscopy structure of ErdS revealed an unprecedented sterol-binding pocket. In addition, the structures in complex with non-hydrolyzable Asp-N-tRNA Asp uncovered a tRNA-guided intramolecular aminoacyl transfer mechanism between two functional domains of the enzyme. The CCA end of tRNA Asp undergoes a large displacement to reach the aa-tRNA transfer active site, while the tRNA elbow is clamped by a long extension of the N-terminal α-helix. The present structural and mutational analyses demonstrate that domain fusion, dynamic repositioning, and tRNA-mediated substrate handover underlie the multifunctional catalytic efficiency of ErdS and facilitate Erg-Asp synthesis independently from protein synthesis. These findings elucidate the unique regulatory mechanism of tRNA-dependent sterol modification and provide insights into fungal membrane dynamics, highlighting potential novel targets for antifungal therapies. Biological sciences/Structural biology/Electron microscopy/Cryoelectron microscopy Biological sciences/Cell biology/Mechanisms of disease Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Aminoacylated transfer RNAs (aa-tRNAs) play an essential role in delivering amino acids (aa) to translating ribosomes. Recent studies have shown that aa-tRNAs also serve as a source of activated aa in a wide range of other cellular processes unrelated to protein synthesis 1 – 5 . In these alternate routes, the tRNA-esterified aa moiety is transferred onto hydroxyl- or amino- groups of diverse acceptor molecules by enzymes called aa-tRNA transferases (ATTs). One structural family of these ATTs shares a common double Gcn5- N -acetyltransferase ( d GNAT)-like fold, and participates in a variety of antibiotic resistance and pathogenicity pathways via aminoacylations of peptidoglycan and glycerolipids in bacteria 6 – 10 . In higher fungi—including human and plant pathogens—we recently identified two new d GNAT-related ATTs, named ergosteryl-3β- O -(L)-amino acid (Erg-aa) synthases (ErxS), which aa-tRNA-dependently conjugate aa to sterols 5 , 11 . The first discovered was the ergosteryl-3β- O -L-aspartate synthase (ErdS), which catalyzes the aspartylation of the 3β-OH group of ergosterol (Erg) 11 – 13 , yielding ergosteryl-3β- O -L-aspartate (Erg-Asp; Fig. 1 A). Concomitantly, we found an Erg-Asp hydrolase (ErdH) that deacylates Erg-Asp back to Erg and aspartate (Asp) 1 . This latter enzyme is an ergosteryl-3β- O -glycine (Erg-Gly) synthase (ErgS), and only found in ascomycota. ErdS is a bifunctional enzyme with unique structural features: the aspartyl-tRNA synthetase (AspRS) domain first activates Asp through adenylation and esterifies it to tRNA Asp , while the appended ATT domain 10 , 14 – 16 transfers the aspartyl moiety from Asp-tRNA Asp to the 3β-OH group of Erg 16 , 17 . Deletion of the erdS gene in Aspergillus fumigatus ( Afm ) and Aspergillus oryzae ( Aor ) had no effect on growth, suggesting that the Asp-tRNA Asp synthesized by ErdS does not significantly contribute to normal biological activities such as protein synthesis, which utilizes a different, classical AspRS 5 , 11 . This bi-functional reaction reminds us of a mechanism observed in other aa-tRNA utilizing enzymes in which, even after aspartylation, tRNA Asp remains bound to the AspRS domain of ErdS, with only the tRNA acceptor arm shuttling directly into the ATT active site without release 18 , 19 . This channeling mode may help fungi produce Erg-Asp efficiently and independently from protein synthesis, but its molecular details remain unknown. Importantly, ErdS is phylogenetically related to bacterial aaPGSs 5 , 11 , but aminoacylates sterols instead of glycerolipids, implying that the d GNAT module of ErdS contains an undescribed sterol-recognition fold. In fungi, Erg plays a crucial role in maintaining the structure and function of cell membranes, thereby directly affecting fungal viability and pathogenicity 20 , 21 . Aspartylation of Erg by ErdS alters its physical and chemical properties, which might affect membranes and modify the responsiveness of cells to external stress, including to antimicrobials 16 , 22 , 23 . However, due to its recent discovery, the functions of Erg-aa in fungi remain elusive. We recently determined that Erg-Asp influences the regulation of asexual sporulation (conidiation) in Aspergillus oryzae ( Aor ), whereas Erg-Gly seems to promote the production of aerial hyphae 24 . Both Erg-aa slightly modify the resistance of Aor towards two antifungal drugs, suggesting a direct influence on membrane properties and/or indirect regulatory effects. ErdS is also present in a plethora of pathogenic fungi, such as Afm , which can cause aspergillosis in immuno-compromised individuals—a life threatening condition associated with poor prognosis 25 —and Magnaporthe oryzae ( Mor ) (rice blast fungus), a cereal pathogen that threatens global agriculture 26 , but the influence of Erg-Asp on their physiology remains unexplored. Here, we show that Erg-Asp participates in asexual sporulation in Afm and Mor . In Afm , the deletion of the erdS gene alters the germination rate of conidia, whereas its deregulation or overexpression increases the production of aerial hyphae and delays conidiation. ErdS and the Erg-Asp hydrolase, ErdH, seem to be expressed at all stages of the Afm life cycle and dynamically localized over the course of fungal development. The deletion of erdS also affects the resistance to high salt stress and the cell wall-targeting compound Congo Red, suggesting that Erg-Asp might participate in a vast array of processes. In this context, and because ErdS presents unique structural features, deciphering the molecular underpinnings of these tRNA-mediated modifications would attract both therapeutic and agricultural interest 27 , 28 . Using cryo-electron microscopy (cryo-EM), we determined the structures of the Afm ErdS apoenzyme and its complex with tRNA. The structures reveal a novel sterol binding pocket and uncover a unique mechanism in which the AspRS long N-terminal α-helix sequesters tRNA Asp , thereby facilitating Asp channeling between the AspRS and ATT active sites, with the RNA moiety acting in a manner mimicking swinging-arm prosthetic cofactors. This mode of tRNA capture might facilitate Erg-Asp synthesis at appropriate locations in a context where ErdS is dynamically localized during fungal development Results ErdS and ErdH are involved in asexual sporulation To obtain insights into the physiological influence of Erg-Asp, we employed two fungal models: the human opportunistic pathogen Afm and the plant pathogen Mor . We used the previously described ∆erdS mutant 1 of Afm , which does not produce Erg-Asp. Despite our best efforts, we could not obtain a Δ erdH mutant of Afm that accumulated Erg-Asp. We circumvented this issue by reintroducing the erdS gene at its locus under the control of the xylose promoter (P Xyl ) in the ∆erdS strain ( ∆erdS ::P Xyl -erdS strain). We also constructed a WT strain in which the P Xyl -erdS construct directly replaced the original erdS copy (WT::P Xyl -erdS strain). Erg-Asp strongly accumulates in those strains when P Xyl -erdS is induced with xylose (Xyl) and is barely synthesized under repressive conditions in the presence of glucose (Glc) ( Fig. S1 A ), confirming the deregulation of ErdS expression. When Afm conidia (asexual spores) are point-inoculated on plates, they form round-shaped filamentous colonies with green conidia in the oldest areas, and the maturing and actively growing mycelia form a thin white circular margin ( Fig. S1 B and S1C ). On rich medium (malt agar), colonies of WT and Δ erdS strains showed no obvious morphological differences, whereas both P Xyl -erdS strains ( ∆erdS ::P Xyl -erdS and WT::P Xyl -erdS ) presented a strong “fluffy” phenotype—dense aerial hyphae— under either repressing or inducing conditions (Fig. 1 B- 1 D), with much larger white margins and smaller conidiation areas. Colonies of all strains presented comparable diameters (Fig. 1 B), suggesting that the larger margins reflected a delay in conidiation. Since rich media can interfere with repression or induction of the P Xyl promoter, we point-inoculated conidia on minimal media (MM) supplemented with or without Glc or Xyl (Fig. 1 C, Fig. S1 D ). Both P Xyl -erdS strains again presented a strong fluffy phenotype under repressing (Glc) conditions, which was reduced—although still visible—under inducing conditions (Xyl), when ErdS was over-expressed. The P Xyl -erdS strains also presented far fewer conidiophores than WT or Δ erdS on the colony edges under repressing conditions, as observed by magnification (Fig. 2 C, 3 rd row, Fig. S1 D ). We next spread conidia of the four strains on MM (Fig. 1 D) and observed the resulting mycelia lawns. The P Xyl -erdS strains presented the fluffy phenotype at 20 h under either inducing or repressing conditions, and in contrast to the WT and Δ erdS strains, no obvious conidiation (greenish-colored mycelium) was visible with the naked eye. Magnification confirmed that the WT and Δ erdS strains produced a high density of conidiophores, while the P Xyl -erdS strains formed very few (Fig. 1 D, rows 1 and 2). As in rich media, the induction of erdS expression (Xyl) did not seem to reverse these phenotypes, likely because the density of mycelia from the convergence of thousands of micro-colonies in lawns exacerbates the remaining fluffy morphology. After 48 h (Fig. 1 D, 3 rd and 4th rows), conidiation eventually occurred in the P Xyl -erdS strains, but the conidiophores were embedded in fluffy aerial hyphae ( Fig. S1 E ). This confirmed that changing the regulation of erdS by placing it under the control of another promoter leads to delayed conidiation. In Mor , we could delete erdS ( ∆erdS , no Erg-Asp synthesis) and erdH ( ∆erdH , no Erg-Asp hydrolase activity) ( Fig. S1 F ). We noted that the WT strain produced undetectable levels of Erg-Asp (Fig. 1 E, left panel ), as we had also observed in Neurospora crassa ( Ncr ) 1 (Fig. 1 E, right panel ), another Sordariomycete. As in Ncr , the deletion of erdH resulted in Erg-Asp accumulation, although at low levels (Fig. 1 E). Interestingly, in contrast to Afm , the deletion of erdS triggered a one order of magnitude decrease in conidiation (Fig. 1 F), which parallels the phenotype observed in Aor for the same mutant 24 . Colonies of both Mor mutants presented a larger white mycelium margin, although it was less prominent than those observed in Afm P Xyl -erdS strains, also reflecting delayed conidiation throughout colony development. Surprisingly, the erdH deletion fully phenocopied that of erdS (Fig. 1 F), suggesting that appropriately regulated levels of Erg-Asp are not only required for proper conidiation timing and/or levels in Mor as well as in Afm , but also for normal hyphal growth in the latter. Erg-Asp influences germination rate Beyond hyphal growth and conidiation, another important aspect of Afm physiology is the germination of conidia. We measured the germination kinetics of WT, Δ erdS , and ∆erdS ::P Xyl -erdS conidia (Fig. 2 A) and observed that the initial isotropic swelling proceeded normally in all cases, with normal proportions of swollen spores in the three strains. However, the emergence of germ tubes was slower with the ∆erdS swollen conidia (mean rate 26.0% h − 1 ) compared to WT conidia (49.5% h − 1 ). As a result, at 9 h, most WT conidia had advanced, as expected, to hyphal extension, while ~ 25% of Δ erdS conidia remained non-germinated. For ∆erdS ::P Xyl -erdS conidia, germination started only after 6 hours (3–4 h for WT conidia) post-inoculation, and the germination rate could not be evaluated under these conditions. This nevertheless suggested that the lack of Erg-Asp synthesis either delays the germination of Afm conidia or alters synchronous germination 29 , 30 . Other types of sterol conjugations are known to impact the immune recognition of fungal spores 31 . We thus decided to monitor the phagocytosis of FITC-stained dormant or swollen conidia by IC-21 murine peritoneal macrophages (IC-21 MPM), using fluorescence microscopy ( Fig. S2 A ). We did not observe significant differences in adhesion to or internalization by macrophages between Afm WT, ∆ erdS or ∆erdS ::P Xyl -erdS conidia, suggesting that Erg-Asp has no major role in the recognition by these immune cells. The same conclusion was drawn from Nanolive microscopy ( Fig. S2 B , Extended movies 1 and 2 ). However, the Kaplan-Meier curves representing the proportion of non-germinated conidia (Fig. 2 B) confirmed that the proportion of non-germinated conidia dropped sharply before 6 h with the WT strain (Multiplicities of infection [MOIs] of 5 and 10 showed no significant difference, p = 0.32, see Fig. S2 B for statistics), with > 80% of germinated spores at 7–8 h post-inoculation (6–7 h after recording started). However, the Δ erdS conidia exhibited slower germination rates at an MOI of 5, and especially at an MOI of 10 (Fig. 2 B). In addition, the final proportion of germinated conidia reached ~ 60% (MOI of 5) or only ~ 30% (MOI of 10) for ∆erdS conidia. This indicated that either the germination success was lower for the mutant, and/or the MOI had an influence on germination. Higher conidial densities are known to decrease germination rates 32 , but here the Δ erdS mutation or Erg-Asp synthesis deregulation ( ∆erdS ::P Xyl -erdS ) might amplify this phenomenon, or perturb synchronous germination within the population. Dynamic localizations of ErdS and ErdH in A. fumigatus Given that ErdS and ErdH—and thus Erg-Asp levels—seem to impact conidiation, germination, and hyphal growth, we analyzed their subcellular localizations throughout the fungal life cycle (Fig. 2 C). We thus replaced the erdH—erdS bigenic cluster of Afm with a version enabling the expression of ErdS and ErdH C-terminally fused to eGFP and mCherry, respectively. Fluorescence microscopy showed that that Afm ErdS and ErdH are both expressed at all stages of the fungal life cycle and already present in resting spores—consistent with the observation of Erg-Asp in total lipids extracted from dormant conidia ( Fig. S2 C ). In resting conidia, ErdS-eGFP is diffusely localized in the cytosol, while ErdH-mCherry is concentrated in cytosolic round-shaped structures that only partially colocalize with ErdS-eGFP (Fig. 2 C, t = 0). In swollen conidia ( t = 3 h), a portion of the ErdS-eGFP starts to concentrate in puncta-like patches that colocalize with ErdH-mCherry puncta. In germinating conidia ( t = 6 h) most of the ErdS-eGFP and ErdH-mCherry signals display granular distribution patterns that, on average, do not seem to colocalize. ErdS-eGFP seems evenly distributed and diffuse in the cytosol of growing mycelia, while ErdH is enriched on or within structures that resemble vacuoles (Fig. 2 C, t = 16 h); however, fractions of both enzymes also appear to colocalize at the plasma membrane, where most of the cellular aspartylable Erg resides. This localization is consistent with previous subcellular fractionation experiments on mycelia 11 . Finally, ErdS-eGFP and ErdH-mCherry are highly enriched at the plasma membranes of remnant germinating structures (Fig. 2 C, t = 16 h). These observations indicate that the localizations of ErdS and ErdH are highly dynamic throughout the fungal life cycle. It is therefore probable that Erg-Asp production temporally and spatially changes throughout the fungal lifecycle, and thus most likely regionally within colonies. Erg-Asp might contribute to stress tolerance and cell wall integrity Since both ErdS and ErdH colocalize at some point in fungal development to the plasma membrane, this prompted us to test whether Erg-Asp could influence the sensitivity of Afm to the cell wall disrupting agent Congo Red (CR). As shown in Fig. 2 D, the Δ erdS strain was more sensitive to CR than the WT. This could indicate that the permeability of either the cell wall or plasma membrane 33 is affected in this mutant and that Erg-Asp might contribute to cell wall and/or plasma membrane integrity, at least under stressful conditions. Interestingly, the ∆erdS ::P Xyl -erdS strain partially recovered CR resistance on MM medium with glucose. In the absence of P Xyl -erdS induction, the variability observed in complementation across replicates likely reflects the leakage due to the presence of the natural P Erd promoter upstream of P Xyl in our construct. However, this will require a more in-depth characterization that goes beyond the scope of this study. In contrast, osmotic stress did not produce significant growth differences (Fig. 2 E), but under high salt stress (NaCl and KCl), the WT strain presented conidiation defects ( Fig. S2 D ), whereas the Δ erdS strain was less affected. The regulation of Erg-Asp synthesis and its levels under various conditions is currently unknown. The only condition under which we found a significant variation of Erg-Asp levels was when the WT Afm strain, first grown in liquid MM containing a nitrogen (N) source (ammonium) was transferred for 5 h into nitrogen-free MM medium, which led to a ~ 1.9-fold decrease in Erg-Asp within the total lipids, as judged by TLC ( Fig. S2 E ), suggesting that N availability might be a signal for Erg-Asp synthesis. However, we did not detect significant differences in growth rates between WT and Δ erdS strains when grown in the presence of various N sources ( Fig. S2 F ) (ammonium, urea, bovine serum albumin, amino acids). When the N source was progressively reduced, however, the ∆erdS and ∆erdS ::P Xyl -erdS strains showed ~ 1.70-fold increased growth rates under N deprivation (0 mM ammonium) or limitation (1 mM), compared to N replete (10 mM) conditions, that the WT strain did not exhibit. These differences tended to decrease under inducing conditions (MM + Xyl) in the ∆erdS ::P Xyl -erdS strain, suggesting the influence of Erg-Asp in this phenotype ( Fig. S2 G ). Given the diversity of effects induced by the absence and over-expression of ErdS in fungi, it seems that the temporally and spatially controlled synthesis of Erg-Asp is required for normal fungal development and propagation. This raised questions regarding how ErdS recognizes Erg and ensures proper Erg-Asp synthesis for normal germination, growth and conidiation, as well as when the fungus copes with stress. We thus performed a structural characterization of ErdS to better understand Erg-Asp synthesis in Dikarya. Structure and architecture of Afm ErdS: the aa-tRNA binding site The closest homologs of ErdS in the d GNATα (+) ATT family are the bacterial aaPGSs that aminoacylate glycerolipids, which suggests that their lipid-binding pockets—which partly reside within the GNAT I subdomain—should differ structurally. No known sterol-binding domain was detected in ErdS and, to our knowledge, no GNAT-related domain characterized thus-far has been reported to bind sterols 34 . Similar to the other ATTs of the same structural fold, the GNAT II domain is expected to recognize the aa-tRNA moiety. Lastly, the mechanism by which the tRNA Asp acceptor stem travels between the AspRS and ATT active sites remains unknown. To identify the aa-tRNA and sterol binding pockets of ErdS, we expressed and purified an Afm ErdS mutant lacking the N-terminal 86 residues (ErdSΔN1-86) for more efficient expression, mixed it with in vitro -transcribed Afm tRNA Asp and/or ergosterol, and performed single particle cryo-EM analyses. The first cryo-EM density map obtained showed that ErdSΔN1-86 bound neither tRNA Asp nor Erg, and that the apo-protein formed an homotetramer ( Fig. S3 ). This tetramer is composed of two ErdS dimers (Fig. 3 A) formed by the interactions between two AspRS domains, in a manner similar to that observed in the crystal structure of yeast AspRS 15 . Despite several attempts to obtain the structure of ErdS with bound Erg, we have not captured the sterol in the ATT domain, possibly due to its low solubility. No information on aa-tRNA or Erg binding could thus be obtained at this stage. Nevertheless, the structures of both AAT domains found in the ErdS dimer could be resolved (Fig. 3 A and 3 B). Each has the d GNATα (+) architecture consisting of two GNAT folds linked by a positively-charged α helix (noted α (+) ) (Fig. 3 B, purple), similar to the architectures of the ATT domain of aaPGSs 10 and the bacterial Fem-ligases (FemX) ( Fig. S4 A ) involved in the tRNA-dependent aminoacylation of the peptidoglycan precursor 8 . In the ErdS dimer, each ATT features a deep, large pocket in the GNAT II domain, next to the α (+) helix, with a surface predominantly covered in positively-charged or neutral residues ( Fig. S4 B ), which likely corresponds to the Asp-tRNA-binding site. As expected, superimposition of the structure of the ErdS ATT domain onto that of FemX, complexed with a tRNA CCA-end analog 9 ( Fig. S4 C-4E ), revealed that the CCA-end analog fit well at the entrance of this cavity (Fig. 3 C; Fig. S4 A-4E ). Moreover, in ErdS, W785 appears in the proximity of the A76 and C75 phosphate groups, with which it could interact via anion-π interactions. R789 is positioned near the ribose of A76, and thus could interact via hydrogen bond interactions (Fig. 3 C). To study the contributions of several residues of ErdS to the Erg-Asp synthesis activity, we constructed the corresponding mutants and expressed the ErdS variants in the yeast Saccharomyces cerevisiae ( Sce )—which possesses no ErxS homolog—as previously reported 11 . The Erg-Asp synthesis was then assessed by thin-layer chromatography (TLC) after lipid extraction. To facilitate comparisons, the Erg-Asp levels were normalized to those of phosphatidylethanolamine (Erg-Asp/PE ratio, noted R ) (Fig. 3 D). Of note, without Erg-Asp, phosphatidylglycerol becomes visible, which results in an R ratio not equal to zero. First, we investigated the conserved GNAT II residues in the putative CCA pocket (Fig. 3 C) and the Lys/Arg residues located in the α (+) helix, which are expected to further bind the tRNA moiety. R749, K750, R752 and R753 were mutated to Ala (ErdSα (0) mutant) or Glu (ErdSα (−) ) (see Fig. 3 B). As expected, ErdSα (−) and ErdSα (0) completely lost the Erg-Asp synthesis activity, with R values (0.12 ± 0.05 and 0.17 ± 0.06, respectively) comparable to that measured for an ErdS ∆ATT mutant lacking the entire ATT domain (0.17 ± 0.06), and well below that of WT ErdS ( R = 1.42 ± 0.27) (Fig. 3 D). We previously observed identical effects when the α (+) helix of the ergosteryl-glycine synthase (ErgS) 5 was mutated. Single mutations in residues shaping the CCA pocket (W785H, W785A, R789A, K790E, G791A, Q793R, H795A, F913A and K916E) dramatically affected the ErdS activity, with all R < 0.5. These results are consistent with their predicted involvement in CCA and tRNA binding (Fig. 3 B and 3 C). Interestingly, the K790E variant ( R = 0.40 ± 0.08) is naturally present in Mor (Fig. 3 D), which might explain the low ErdS activity in this fungus when erdH is deleted (no Erg-Asp hydrolase) (Fig. 1 E). ErdS GNAT I domain harbors a new sterol-binding fold We next docked Erg within a putative sterol-binding cavity, which superimposes with the peptidoglycan-binding pockets of bacterial ATT homologs (Fig. 3 E and 3 F). Erg fits well in this cavity, with a conformation consistent with favorable steric and electrostatic interactions to orient the 3β-OH group towards the terminal A76 of tRNA, to which Asp is esterified. A structural comparison with the PG-bound form of the homolog LysPGS 10 revealed, as expected, that the overall shape of the pocket differs substantially ( Fig. S4 F ), indicating that this predicted sterol-binding pocket (Fig. 3 E and 3 F) is indeed unique in the d GNATα (+) ATT family and might constitute a new sterol-binding fold. Based on a multiple alignment of 98 ErdS orthologs, we selected 16 potentially critical residues within this cavity (Y637, T641, S642, T643, S644, W645, D647, R649, N677, E712, C728, E731, E798, W802, Q836 and K838) among which 13 are strictly or moderately conserved ( Fig. S5 ). The Q836R mutation completely abolished the ErdS activity ( R = 0.14 ± 0.05), while other mutations significantly reduced the Erg-Asp synthesis activity. The S644A mutation had the strongest effect ( R < 0.5), while the W645A, D647R and K838E mutations had milder consequences (0.5 < R < 1.0) (Fig. 3 D). Mutations of the remaining 11 residues had only slight or statistically non-significant effects. Since the ErdS variants were all expressed at comparable levels in Sce strains (Fig. 3 D, immunoblot), the decreased ErdS activities solely originate from the introduced mutations (with exception of F913A, which was expressed at low levels). We ruled out the possible long-range effects of these mutations on the AspRS activity—that would also reduce Erg-Asp synthesis, even if Asp-tRNA can be scavenged from the cellular pool 11 —since all of these ErdS variants could replace the endogenous Sce AspRS (Dps1) by complementation of a dps1∆ strain 35 ( Table S2 ). These results are in good agreement with our predicted location of the sterol-binding domain of ErdS. Sterol aspartylation by ErdS is highly promiscuous We previously observed that Afm ErdS aspartylates cholesterol (Cho) in addition to Erg, despite the structural differences between the two sterols 11 . Consequently, Cho fits well within ErdS’s sterol pocket in docking models ( Fig. S6A ). Diosgenin, which features an unusual cyclized alkylyl side-chain ( Fig. S6A ), also seems to bind productively, suggesting that ErdS might have broad specificity for sterols. To investigate both the ErdS promiscuity towards sterols and the structural determinants for sterol recognition, we performed an in vitro sterol aminoacylation assay 11 with a panel of structurally different sterols (Fig. 3 G and 3 H, Fig. S6B and 6C ). Afm ErdS aspartylates sterols regardless of the alkylyl side-chain’s structure, since in addition to Erg and Cho (Fig. 3 G, left panel , lanes 1, 2 and 4), it aspartylated the human hormones pregnenolone and trans -dehydroandrosterone (DHA), although they present much smaller polar side-chains (Fig. 3 G, left panel , lanes 6 and 7). The lower aspartylation levels suggest that the alkylyl moiety could enhance the proper anchoring within the sterol pocket (Fig. 3 G). Consequently, a variety of other sterols can be used as substrates, despite the fact that they differ in the numbers (0, 1 or 2) and positions of saturations in the B-ring or in the bulkiness, saturation, and methylation pattern of the alkylyl side chain ( Fig. S6B and S6C , lathosterol, zymosterol, β-sistosterol, stigmasterol, coprostanol, fucosterol, 5α-cholestan-3β-ol). As predicted above ( Fig. S6A ), diosgenin is efficiently aspartylated by ErdS, indicating that a cyclized side chain does not prevent recognition and aminoacylation. Lanosterol, which features a 4-dimethylated position in the A-ring of the cyclopentanophenantrene (sterane) nucleus, however, is not aspartylated in vitro (Fig. 3 G, left panel, lane 3, Fig. S6B , lane 3), indicating that such bulky modifications may act as antideterminants for sterol recognition. Similarly, β-estradiol, with an aromatic A-ring, is not a substrate (Fig. 3 G, left panel, lane 8), despite its otherwise common features with DHA, showing that a non-aromatic ring is required. Interestingly, coprostanol and epicoprostanol are identical, except that the first possesses a 3-OH group in the β configuration, whereas the second is in the α configuration. These two steroids and 5α-cholestan-3β-ol have the same alkylyl side chain as Cho (but no unsaturation at position 5 in the steran nucleus). As expected, coprostanol and 5α-cholestan-3β-ol are aspartylated at similar levels as Cho, despite the absence of the Δ5 unsaturation and their different absolute configurations at position C5 (Fig. 3 G, right panel ). In contrast, epicoprostanol is not aminoacylated, most likely because the 3α-OH group is oriented away from the catalytic residues (see below) and the aminoacylated A76 residue of tRNA Asp . Consequently, cholic acid, which has a 3α-OH group, is also not a substrate (Fig. 3 G, left panel , lane 5). This promiscuous activity also exists in vivo , as demonstrated by the heterologous expression of Afm ErdS in Sce Erg biosynthesis pathway mutants 36 ( erg3 ∆, erg4 ∆, erg5 ∆ and erg6 ∆), which accumulate various Erg intermediates ( Fig. S6D and S6E ). TLC analyses of total lipids from these strains revealed that ErdS aspartylates Erg intermediates that mostly differ in the saturation of the B-ring and the alkylyl side-chain, as expected from our in vitro results; notably, the erg6 Δ strain accumulates zymosterol, which was also aminoacylated by ErdS in vitro ( Fig. S6B , lane 5). Therefore, ErdS is highly promiscuous for sterols, with the minimal requirements that the A-ring is non-aromatic and free of methyl groups and the 3-OH is in the β configuration. Organization of the active site and catalytic mechanism of transesterification Simultaneous docking of Erg and the CCA analog revealed that the 3-OH group of Erg, when in the β configuration, is oriented towards the 2´-OH of the ribose moiety of A76, in a geometry suitable for transesterification (Fig. 3 E and Fig. S6F-S6G ). The α configuration evidently misorients the 3-OH group of Erg (or any sterol) within the active site, preventing it from reacting with the Asp-tRNA Asp ester bond. In FemX complexed to the CCA analog and a peptidoglycan precursor, the nucleophile is provided by the ε-amino group of a Lys side chain from the peptidoglycan precursor before transpeptidation. By contrast, Erg must first be activated by ErdS. We propose ( Fig. S6F and S6G ) that H795 acts as a general base to deprotonate the 3-OH group of Erg, thereby generating a nucleophilic alkoxide. This activated 3-alkoxide is positioned to attack the carbonyl carbon of the aminoacyl ester in Asp-tRNA Asp . During this reaction, the negative charge developing on the carbonyl oxygen is likely stabilized by the positively-charged guanidinium group of R914, facilitating the transesterification ( Fig. S6G ). The K305 residue of FemX has an equivalent function, and is located at the same position in superimposed models. This mechanism provides hints toward elucidating how the active site architecture of ErdS supports the selective transfer of the aspartyl group to only 3β-OH-containing sterol substrates. The presence of methyl groups at C4 of the sterol A-ring likely introduces steric hindrance, preventing proper accommodation within the binding pocket. The N-terminal long α-helix of ErdS clamps the tRNA elbow to ensure enzymatic activity The N-terminal extension in the Class IIb aaRSs of higher eukaryotes contains a conserved alpha-helix, which has been suggested to play a crucial role in enhancing their affinity for tRNA 37 – 40 . Although the crystal structure of AspRS has been determined, this N-terminal region was deleted in the resolved structure, and thus requires further investigation to elucidate its role in tRNA binding 15 , 41 . Notably, ErdS also possesses a similar N-terminal helix that includes the conserved xSKxxLKKxx motif (with the exception of Mor ), which was previously proposed to be critical for tRNA binding 39 , 40 (Fig. 4 A). The recently reported cryo-EM structure of human LysRS 42 provides insights into the partial structure of this helix and its interactions with tRNA. However, the density corresponding to the anticodon-binding region remains unresolved, and its complete structural configuration awaits elucidation. To investigate the role of the N-terminal helix, we prepared full length ErdS (FL-ErdS), incubated it with in vitro- transcribed Afm tRNA Asp , and performed a cryo-EM analysis. The structure revealed the dimeric configuration of ErdS in complex with two tRNA Asp molecules (Fig. 4 B; Figs. S8 and S9 ). In the complex, each protomer of the ErdS dimer binds one tRNA Asp molecule (Fig. 4 B- 4 D). The anticodon of tRNA Asp interacts with the anticodon-binding domain (ABD) of the AspRS domain, and the acceptor stem is positioned within the catalytic domain of AspRS, consistent with the previously reported crystal structure of the Sce AspRS/tRNA Asp complex 15 . The ATT domain was disordered, suggesting that it moves flexibly relative to AspRS. Interestingly, residues 59–102 of the NTD form an extended linear pair of α-helices separated by a connective loop, which spans from the anticodon to the elbow region of tRNA Asp (Fig. 4 B- 4 E). This N-terminal region is rich in polar amino acids, which form multiple interactions with the phosphate backbone, ribose moieties, and nucleotide bases of tRNA Asp . Residues R61 and N62 interact with the phosphate backbone of residues G50 to G52 in the T-stem. The highly conserved K65 interacts with the ribose of G57 in the T-loop. C20a of tRNA Asp is flipped outward, with its base forming a salt bridge with R69 of the NTD. Lastly, E70 and S77 interact with the phosphate backbone of tRNA Asp ’s variable loop. Further stabilization is provided by E81, Q84, and D87 within the connective loop between the α-helices, which, along with H92, interact with either the ribose or the phosphate backbone of the anticodon stem. We previously reported that full-length Afm ErdS can complement an Sce dps1 Δ strain (AspRS gene deletion), although less efficiently than an Afm ErdS ΔATT variant in which the ATT domain was removed, likely because Sce tRNA Asp was used primarily for Erg-Asp synthesis over protein synthesis in this context, which lacked extra AspRS 11 . Based on our cryo-EM structure, we hypothesized that the NTD could also contribute to tRNA Asp sequestering within ErdS, to ensure its dedicated participation in Erg-Asp synthesis. To ascertain the contribution of the observed N-terminal domain in tRNA Asp sequestering, we designed mutants with progressive NTD truncations (ErdSΔN30, ΔN60, and ΔN84, in which the first 30, 60 or 84 residues were removed, respectively) and tested their complementation efficiency in the dps1 Δ strain by plasmid shuffling. First, we confirmed that the ErdS ΔATT mutant complemented the dps1 Δ strain as efficiently as Dps1 ( Sce AspRS). Strikingly, the growth progressively improved with the NTD truncations of Afm ErdS, with the ΔN84 mutant behaving like the ErdS ΔATT mutant, as judged by the Sce colony sizes (Fig. 4 F) and by drop tests ( Fig. S7 ). This indicates that the region between residues 60 and 84— i.e. , where the xSKxxLKKxx motif lies—has the strongest contribution to growth reduction, and likely to tRNA Asp sequestering. The growth improvement was interpreted as the easier release of Asp-tRNA Asp from ErdS, in agreement with structural results. In parallel, we visualized Erg-Asp production in those strains and observed that Erg-Asp synthesis dropped by 17.8-, 15.0-, and 12.1-fold, respectively, for the ErdSΔN30, ΔN60, and ΔN84 mutants, indicating that the efficiency of Erg-Asp synthesis is affected by even the smallest truncation (Fig. 4 F), and showing that the NTD is crucial for the full activity. In the case of Aor ErdS, the complementation of the dps1 Δ strain was highly inefficient (Fig. 4 G), but the truncation of the NTD segment down to residue 90 (equivalent to Δ84N in Afm ErdS) restored WT growth. Again, Erg-Asp synthesis was reduced (2.9-fold) in this mutant, compared to the full-length enzyme. These results suggest that, apart from the ATT domain, the NTD significantly contributes to tRNA Asp utilization in the in vivo context, likely through sequestering it within ErdS, making it unavailable for protein synthesis and thereby reducing growth, while strongly increasing the efficiency of Erg-Asp synthesis. This explains why all ErdS-containing fungi have a second, separated AspRS 5 , 11 (ref PNAS and Ref JBC) that ensures a sufficient supply of Asp-tRNA Asp for protein synthesis. tRNA behaves like a prosthetic swinging arm during the multi-enzymatic process of ErdS The cryo-EM structural analysis of the ErdS/tRNA Asp complex revealed 3D classes with density suggestive of the ATT domain. Further analysis using a mask covering these weak densities improved the map, confirming that they extended from the C-termini of both AspRS domains, which validated them as the ATT domain (Figs. 5 A, 5 B and S8C). In one ErdS protomer (ErdS1), the ATT1 module is positioned adjacent to the elbow of the tRNA 1 Asp bound to the corresponding AspRS1 domain, on the same side where the NTD1 also interacts along the anticodon stem and the elbow region. Interestingly, tRNA 1 Asp is not only supported at the elbow region by the ATT1 domain and the NTD1, but its CCA end is located near the ATT2 module from the other protomer (ErdS2), effectively sandwiching tRNA 1 Asp (Fig. 5 A). This explains why the dps1 Δ strain expressing WT Afm ErdS grew more slowly than the strains expressing Dps1 ( Sce AspRS) or the Afm ErdS ΔATT mutant (Fig. 4 F). ErdS predominantly directs Asp-tRNA Asp towards Erg-Asp synthesis—resulting in slower growth in the absence of an independent AspRS—because both the NTD and ATTs prevent its aminoacylated tRNA Asp from being released. In light of this structural information, we propose that the ATT domains of ErdS dimers cooperate not only in Erg-Asp synthesis but also, together with the NTD, in sequestering tRNA Asp . To further investigate the interaction between the ATT domain and the CCA end of tRNA Asp , we attempted the cryo-EM structural analysis of ErdS bound to Asp-tRNA. We reasoned that the ATT domain requires the Asp moiety to bind tRNA Asp over the AspRS domain, which in contrast recognizes uncharged tRNA Asp . Since aa-tRNAs are unstable, we prepared a non-hydrolyzable Asp-tRNA Asp analog (Asp- N -tRNA Asp ), by using a modified tRNA Asp transcript in which the 3´-hydroxy group of the terminal ribose is replaced by a 3´-amino group, so that the ester bond between Asp and tRNA Asp is replaced by an amido bond upon Flexizyme-mediated in vitro aspartylation 43 – 45 . We also prepared an ErdS variant with nine mutations in the AspRS domain (Q334E, S335A, N362P, S363A, N364P, H366A, R367A, H368A, and Y494F) to reduce its tRNA binding affinity and allow the Asp-CCA end to enter the ATT catalytic site. We then incubated Asp- N -tRNA Asp with this purified ErdS variant and performed a cryo-EM structural analysis. This revealed the structure of an ErdS dimer bound with only one Asp- N -tRNA Asp (Fig. 5 B, Fig. S10 ). This Asp- N -tRNA 1 Asp was bound to the ABD of AspRS1 in a manner identical to that in the WT ErdS/tRNA Asp complex (Fig. 4 B and Fig. 5 A). Surprisingly, in this case the CCA end protruded away from the AspRS1 active site (Fig. 5 B to 5 E, Fig. S11 ). This structural rearrangement is accompanied by a 23 Å movement of the tRNA Asp CCA end and an 8 Å shift of the tRNA elbow (Fig. 5 C). The ATT1 domain-binding Asp- N -tRNA Asp (Fig. 5 B) was positioned similarly to that observed in the previous ErdS/tRNA Asp complex (Fig. 5 A). It also interacted with the elbow of tRNA Asp , whereas the ATT2 domain from the tRNA Asp -free protomer (ATT2) showed no detectable density. Surprisingly, these orientations of the acceptor stem and the CCA end of Asp- N -tRNA 1 Asp did not direct them towards the active site (CCA pocket) of ATT1, raising questions on the mechanism by which the Asp moiety could be used to modify Erg. We aligned the tRNA Asp -free AspRS1 domain with the AspRS domain of the ErdS/tRNA Asp complex characterized previously, in order to determine the positions of the missing tRNA 2 Asp and the ATT 2 module. These models revealed that the CCA end of Asp- N -tRNA 1 Asp was even closer to the ATT2 domain (Fig. 5 D). Notably, the α (+) helix of ATT2, which was predicted to interact with the tRNA Asp acceptor arm, was the closest to the CCA end (Fig. 5 E). The W785 and K916 residues, which are crucial for the CCA interaction and the activity (Fig. 3 A to 3 D), were also close to the CCA end. These results suggest that, in the ErdS dimer, after aspartylation by the AspRS1 domain, the CCA end of tRNA 1 Asp swings to the ATT2 domain of the other protomer, allowing the efficient aminoacylation of Erg (Fig. 6 ). Since tRNA Asp seems to be trapped by its interactions with the NTD and both ATT domains, it is likely that it does not dissociate from ErdS, and that the acceptor arm shuttles back and forth from one active site (AspRS1) to the next (ATT2), serving as a prosthetic swinging arm that shuttles an activated Asp moiety for Erg-Asp synthesis. This fully explains the low capacity of ErdS to replace the endogenous AspRS in Sce , unless either the ATT or NTD domain is removed to facilitate Asp-tRNA Asp release from ErdS to promote protein synthesis. Discussion Our study reveals a distinct mechanism by which an aa-tRNA is employed for small-molecule aminoacylation in eukaryotes. Through structural and biochemical analyses, we show how ErdS catalyzes the transfer of aspartate from Asp-tRNA Asp to the 3β-OH group of Erg—a lipid central to fungal membrane physiology. This reaction is enabled by a previously uncharacterized sterol-binding pocket within the ATT domain and a tRNA-mediated handover mechanism that coordinates two spatially separate active sites. These findings provide new insights into how enzymes can co-opt aa-tRNAs to expand their catalytic repertoire beyond and independently from translation. ErdS does not rely on the diffusion of intermediates. Instead, it uses the tRNA as a dynamic tether to direct the flow of the aminoacyl (aspartyl) group. First, the RNA moiety of the ErdS/tRNA Asp complex is sequestered within the ribonucleic particle, not only through interactions with the two ATT domains from the two ErdS protomers, but also by a long NTD that further interacts with the anticodon and elbow regions. This reinforces the interaction of the molecule with the AspRS part, thus stabilizing the tRNA through extensive interactions and guiding its movement across the enzyme, which likely prevents its release upon aspartylation, as suggested by complementation experiments in Sce . The acceptor arm and CCA end of tRNA Asp can thus undergo a large-scale repositioning (~ 23 Å) to reach the ATT domain, while the domain itself is flexibly rearranged to facilitate this interaction. Unexpectedly, in the ErdS protomer, the tRNA acceptor arm does not simply shuttle from the AspRS active site to its appended ( cis ) ATT domain, as we initially thought (Fig. 1 A). Instead, the cis ATT functions as a stabilizer of the ErdS/tRNA Asp interaction, whereas the second ATT domain, situated in the second ErdS protomer, captures the CCA-end of tRNA Asp through interactions with the α (+) helix, and functions as a trans -acting enzyme, using the aspartyl moiety to transfer it onto Erg. In this context, the tRNA functions as an architectural bridge—dynamically connecting the AspRS and ATT domains—in a manner reminiscent of the swinging arms seen in multienzyme complexes 46 and of the shuttles between aminoacylation and amino acid editing functions observed in aminoacyl-tRNA synthetases 47 , 48 Schematics of this molecular choreography are depicted in Fig. 6 and Extended Data Movie 3 . The domain architecture of ErdS, which fuses AspRS with a d GNAT-type ATT module, is essential for this coordinated reaction. Our structural and mutational analyses suggest that the extended NTD, which differs from those found in canonical cytoplasmic AspRSs, plays a critical role in clamping the tRNA to stably position it for productive handover. Shortening of the NTD reduces the Erg-Asp synthesis efficiency in vivo in the yeast heterologous model, and we previously observed that separating the AspRS and ATT domains also reduces the activity, including when the ATT domain is expressed alone in Aor 11 , illustrating the catalytic advantages of a single bifunctional enzyme. This integration of synthetase and transferase activities into a single polypeptide represents a functional innovation, enabling direct control over substrate transfer and reactivity. Our complementation experiments in Sce illustrated that ErdS poorly releases Asp-tRNA Asp , which is thus efficiently trapped throughout the Erg-Asp synthesis cycle, leading to decreased protein synthesis. Hence, in nature, Dikarya harboring ErdS always possess a second, canonical AspRS to ensure efficient protein synthesis 11 . Sequestering of tRNA Asp within ErdS also provides an advantage by diverting a subset of tRNA Asp from the cytosolic pool and dedicating it solely to Erg-Asp synthesis. This differs from free-standing ATTs, such as the Erg-Gly synthase also present in fungi, which rely on an independent aa-tRNA synthetase—and therefore on protein synthesis—to produce their aa-tRNA substrate in the context of competition with elongation factors fueling translating ribosomes with aa-tRNAs. The fusion between the AspRS and ATT domains in ErdS and the molecular mechanism described above may ensure proper Erg-Asp levels under challenging conditions and at dedicated subcellular localizations, to enable proper mycelium development, conidiation, and likely cell wall integrity, independently from protein synthesis. The deletion of erdS in Afm , Aor , or Mor leads to contrasting phenotypes. Coniditation is strongly reduced in Δ erdS mutants of Aor 24 or Mor (this study), but not in Afm . This may reflect differences in regulatory circuits between species or in the—direct or indirect—contribution of Erg-Asp to such processes. Of note, in the Δ erdS strain of Afm , the ergS gene that encodes the Erg-Gly synthase (ErgS) is still present, and we cannot exclude the possibility that Erg-Gly has overlapping functions that could attenuate phenotypes. Conversely, Mor lacks ErgS. Moreover, the deletion of the Erg-Asp hydrolase gene erdH phenocopies the Δ erdS mutation in Mor , suggesting that the deregulation of Erg-Asp levels, rather than the absence of ErgS, plays significant roles in phenotypes. Nevertheless, the deregulation or impaired synthesis of Erg-Asp always affects conidiation to various degrees—from decreased conidia production to differences in conidiation timing. In Afm it also affects vegetative growth (aerial hyphae production) and spore germination, and likely stress resistance as well. These effects are expected to differ greatly between ErdS/H-containing fungi, as illustrated by the differences in Erg-Asp levels during vegetative growth between Afm and Aor on the one hand (Erg-Asp synthesis) and Ncr and Mor on the other hand (no detectable Erg-Asp). This may also be amplified by the presence or absence of ErgS, and thus, of Erg-Gly. In addition, the spatial and temporal regulations of Erg-Asp synthesis might also differ, not only between fungal species but also at the colony and cell levels within each species. Overall, Erg-Asp synthesis (ErdS) and regulation (ErdH) likely contribute to the fitness of fungi, as supported by their conservation across fungal clades in Dikarya. Further work is needed to understand the contributions of Erg-aa to the pathogenicity and stress resistance of these species, and to clarify their functions in reproduction and growth. The sterol-binding pocket discovered in the ATT domain adopts a geometry and electrostatic environment tailored to accommodate the rigid structure of Erg. The deep, hydrophobic cavity lined with basic residues provides both shape and charge complementarity to the ligand. This binding mode is structurally distinct from lipid-interacting enzymes such as LysPGS, and illustrates how the d GNAT fold can be repurposed for the selective recognition of sterol substrates. Given the essential role of Erg in fungal membrane integrity and drug resistance, this well-defined pocket may serve as a promising scaffold for antifungal drug development. It appears highly promiscuous for sterols, but it is worth mentioning that while Erg is the main sterol of numerous fungi—including Afm , Aor , Mor or Ncr —different types of sterols are used in other species 49 that also possess ErdS, such as the Erg-related sterols in Agaricomycotina and the 24-ethylcholesterol-family molecules in Pucciniomycotina. This apparent promiscuity may reflect this molecular diversity, and could result from ErdS’s evolutionary history within Dikarya. By integrating amino acid activation, tRNA coordination, and sterol modification within a single, spatially organized framework, ErdS exemplifies a catalytic strategy built on architectural precision and dynamic domain interplay. Rather than simply combining functions, ErdS choreographs the positioning of its substrates through tRNA-guided movements and domain rearrangements. This mechanism not only reveals a unique solution to non-ribosomal aminoacyl transfer, but also offers a design principle for engineering synthetic enzymes with programmable spatial control. The structurally resolved sterol-binding pocket further provides a tractable and selective platform with potential relevance to antifungal drug development. Declarations Author information H.M. performed structural analyses with assistance from M.N., Y.K. and Y.I. N.Y., N.M. and S.Z. conducted fungal genetics, phenotypic characterization, and biochemical assays, with assistance from B.S., F.F. and H.D.B. L.S. and F.M. performed the Nanolive imaging experiments. H.B.G. and Y.M.H. prepared the Asp-tRNA Asp analog used for structural analysis. H.M. and F.F. wrote the manuscript with input from all authors. H.D.B. and O.N. supervised the research. Acknowledgements We thank Dr. Y. Goto and Dr. H. Suga for providing the chemically synthesized Asp-DBE substrate. Cryo-EM data were collected at the Cryo-EM facility of The University of Tokyo. This work was supported by the “N-FLAMS” project from the Agence Nationale de la Recherche (ANR-20-CE44-0002), and by the Integrative Molecular and Cellular Biology (IMCBio) program as part of the Interdisciplinary Thematic Institutes (ITI) 2021–2028 initiative of the University of Strasbourg, CNRS, and INSERM, supported by IdEx Unistra (ANR-10-IDEX-0002) and EUR IMCBio (ANR-17-EURE-0023), under the framework of the French Investments for the Future Program (to N.Y., N.M., S.Z., B.S., F.F., and H.D.B.). Additional support was provided by the University of Strasbourg and CNRS (to N.Y., N.M., S.Z., B.S., F.F., and H.D.B.), by JSPS KAKENHI Grant Number 23KJ0722 (to H.M.), and by JST CREST JPMJCR20E2 (to O.N.). References Roy H, Ibba M (2008) RNA-dependent lipid remodeling by bacterial multiple peptide resistance factors. Proc. Natl Acad. Sci. USA 105, 4667–4672 Moutiez M, Belin P, Gondry M (2017) Aminoacyl-tRNA-Utilizing Enzymes in Natural Product Biosynthesis. 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PLoS ONE 5:e10899 Methods Preparation of ErdS for Cryo-EM The gene encoding A. fumigatus ErdS Δ 85 (residues 86-947) was cloned into a pE-SUMO vector. The N-terminally His 6 -SUMO-tagged ErdSDN85 was expressed in Escherichia coli Rosetta2 (DE3). The E. coli cells were cultured at 37 °C until the A 600 reached 0.6, and protein expression was induced by adding 0.2 mM isopropyl β-D-thiogalactopyranoside (NacalaiTesque). The E. coli cells were further cultured at 20 °C overnight, collected by centrifugation, resuspended in lysis buffer (20 mM Tris-HCl, pH 8.0, 300 mM NaCl, 20 mM imidazole, 50 µM TCEP), added 50 µM PMSF and then lysed by sonication. The lysate was centrifuged, and the supernatant was incubated with Ni-NTA Agarose resin (Qiagen) for 1 h. The resin was washed with lysis buffer, and the protein was then eluted with elution buffer (20 mM Tris-HCl, pH 8.0, 300 mM NaCl, 300 mM imidazole, 50 µM TCEP). The eluted protein was treated with the SUMO protease and dialyzed against dialysis buffer (20 mM Tris-HCl, pH 8.0, 50 mM NaCl, 40 mM imidazole, 50 µM TCEP). The protein sample was passed through a Ni-NTA Agarose column, to remove the His 6 -SUMO and SUMO protease. The protein was then loaded onto a 5 mL HiTrap SP HP column (GE Healthcare), equilibrated with buffer (20 mM Tris-HCl, 50 mM NaCl, 50 µM TCEP). The protein was further purified by chromatography on a HiLoad 16/600 Superdex 200 column (GE Healthcare), equilibrated in elution buffer (20 mM Tris-HCl, pH 8.0, 150 mM NaCl, 5 mM MgCl 2 , 50 µM TCEP). The purified ErdSΔ85 was concentrated to 2 mg/mL by ultrafiltration, frozen in liquid nitrogen, and stored at −80°C. The full-length A. fumigatus ErdS (residues 1-947) was also expressed and purified in the same way as ErdSD85. Except that a 50 mM NaCl concentration was used as a buffer and an affinity chromatography column, HiTrap Heparin (GE Healthcare), was used instead of the HiTrap SP HP used during purification. The purified ErdS was concentrated to 3 mg/mL by ultrafiltration, frozen in liquid nitrogen, and stored at −80°C. ErdS variant harboring nine substitutions at the AspRS interaction interfacewas also expressed and purified in the same way as full-length ErdS. Based on our ErdS structure, we identified nine residues in AspRS domain that interact with the tRNA Asp acceptor stem. To weaken this interaction, these residues were mutated as follows: Q334E, S335A, N362P, S363A, N364P, H366A, R367A, H368A, and Y494F. The purified ErdS was concentrated to 3 mg/mL by ultrafiltration, frozen in liquid nitrogen, and stored at −80°C. Preparation of tRNA Asp and Asp- N -tRNA Asp for Cryo-EM The gene encoding A. fumigatus tRNA Asp in which a hammerhead ribozyme sequence and the consensus T7 promoter sequenceon its 5′ end was cloned into a pUC119 vector. tRNA Asp was transcribed in vitro at 37°C for 16-20 h using T7 polymerase, followed by incubation at 60°C for 16-20 h with the addition of MgCl 2 to a final concentration of 30 mM. The target tRNA bands were separated by urea-PAGE, extracted by EluTrap, ethanol precipitated, buffer exchanged, and stored at −80°C. For the preparation of the Asp- N -tRNA Asp , 3′-Overlapping forward and reverse DNA oligonucleotides (Integrated DNA Technologies) were hybridized and enzymatically extended to form a dsDNA template in which a hammerhead ribozyme sequence was sandwiched between the consensus T7 promoter sequence and the coding sequence for Afm tRNA Asp as described 1 . To suppress non-templated transcription, the 5′-end of the reverse oligo contained two 2′-OMe nucleotides. tRNA Asp was transcribed in vitro at 37°C for 6 h using T7 polymerase, followed by incubation at 60-65 °C for 1 h with the addition of MgCl 2 to a final concentration of 30 mM. The target tRNA bands were separated by urea-PAGE, crushed with a glass rod, and extracted into TE by shaking overnight. After centrifugation the supernatant was passed through a 0.45 µm syringe filter (33 mm; Thermo Fisher) and the tRNA was ethanol precipitated. 3′-amino-tailing followed the protocol described in 2 . Briefly, reactions containing 100 µM tRNA Asp , 32 µM human CCA-adding enzyme,1.75 mM 3′-amino-ATP (BioLog Life Science Institute, Bremen, GER), 1 mM sodium pyrophosphate, 10 mM MgCl 2 , 1.0 mM DTT, 0.2 units/µL murine RNase inhibitor (NEB), and 50 mM glycine (pH 9.0) were incubated for 1 hr at 37°C. Prior to workup pyrophosphate was hydrolyzed by adding inorganic pyrophosphatase (0.1 U/µl; New England Biolabs) to the reaction and incubating an additional 15 min at RT. Each reaction was quenched with 2.5 M NaOAc (pH 5.0), extracted with an equal volume of pH 5.0 phenol:chloroform-isoamyl alcohol (80:17:3), and the tRNA ethanol precipitated. The extent of 3′-tailing was determined to be 60% by biotin-streptavidin gel shift assay 3 . Amino-tailed tRNA was stably charged by incubation with flexizyme (dFx) and Asp-DBE 4,5 . Briefly, reactions containing 40 µM tRNA (60% amino-tailed), 31 µM flexizyme, 3 mM Asp-DBE, 375 mM MgCl 2 , 12.5% DMSO, and 0.1 M K-HEPES (pH 7.5) were incubated in a thermomixer at 25 °C with shaking for 72 hr. After 24 and 48 hr each reaction was supplemented with fresh 200 mM Asp-DBE. Reactions were quenched with 0.1 volume 2.5 M NaOAc pH 5.0 followed by ethanol precipitation. The combined pellets were dissolved in 0.1 M glycine pH 9.0 and incubated 3 hrs at 37 °C to hydrolyze alkali-labile aminoacyl ester linkages. After ethanol precipitation tRNA was purified away from flexizyme by urea-PAGE. Since stably charged tRNA could not be resolved from amino-tailed tRNA by gel electrophoresis, mass spectrometry was used to determine the charging efficiency, which proved to be 40%. Cryo-EM sample preparation,data collection and image processing For the ErdS tetramer structure, ErdS Δ85 and tRNA Asp were mixed at a molar ratio of 1:1 and incubated at 37°C for 20 min. The 3 µL of the prepared sample was applied to a freshly glow-discharged Cu/Rh 300 mesh R1.2/1.3 grid (Quantifoil), in a Vitrobot Mark IV (FEI) at 4 °C, with a waiting time of 30 s and a blotting time of 4 s under 100% humidity conditions. The grid was plunge-frozen in liquid ethane cooled at liquid nitrogen temperature. Cryo-EM data were collected using a Titan Krios G3i microscope (Thermo Fisher Scientific), running at 300 kV and equipped with a Gatan Quantum-LS Energy Filter (GIF) and a Gatan K3 Summit direct electron detector in the electron counting mode. Micrographs were recorded at a nominal magnification of 105,000×, corresponding to a calibrated pixel size of 0.83Å, with a total dose of 49 electrons per Å 2 . The data were automatically collected by the image shift method using the SerialEM software, with a defocus range of −1.6 to −0.8 μm, and 3,357 movies were obtained and processed using RELION-3.1 6–10 . From the 3,357 motion-corrected and dose-weighted micrographs, 2,021,179 particles were initially picked, and extracted at a pixel size of 3.32 Å. These particles were subjected to several rounds of 2D and 3D classifications. The selected 240,624 particles were re-extracted at a pixel size of 1.66 Å, and then subjected to 3D refinement, per-particle defocus refinement, beam-tilt refinement, Bayesian polishing, and 3D classification with the mask focusing on ErdS monomer 11 . Finally, a single class of 35,812particles was selected after signal subtraction and symmetry expansion to C 1 , yielding a map at 3.6 Å resolution, according to the Fourier shell correlation (FSC) = 0.143 criterion 12 . As for the ErdS/tRNA Asp complex, the tRNA Asp that had been aspartylated by AspRS was mixed with twice the molar volume of ErdS and ergosterol to a final concentration of 630 µM were incubated at room temperature for 1 h. The sample was added to a previously glow discharged 300 mesh Au R1.2/1.3 grid (Quantifoil) for cryo-EM observation and rapidly frozen. Cryo-EM data were collected using a Titan Krios G4 microscope (Thermo Fisher Scientific), running at 300 kV and equipped with a Gatan Quantum-LS Energy Filter (GIF) and a Gatan K3 Summit direct electron detector in the electron counting mode. Micrographs were recorded at a nominal magnification of 105,000×, corresponding to a calibrated pixel size of 0.83Å, with a total dose of 48 electrons per Å 2 . The data were automatically collected by the image shift method using the EPU software (Thermo Fisher Scientific), with a defocus range of −1.6 to −0.4 μm, and 2,334 movies were obtained and processed using cryoSPARC v4 13 . Initial particle picks were used as templates for repicking using Topaz, yielding 885,089 particles from all micrographs, and extracted 14 . These particles were subjected to 2D classification, and the selected 510,043 particles were combined for multiple rounds of three-dimensional classification (ab initio model generation and heterogeneous refinement). The best class containing 20,590 particles was refined using non-uniform refinement after CTF refinement, yielding a map at 3.66 Å resolution 15 .To see the ATT domain, particles were subjected to several rounds of three-dimensional classification (3D refinement and heterogenous refinement). The best class containing 15,808 particles was refined using Non-uniform Refinement, yielding a map at 3.43 Å resolution. As for the ErdS/tRNA Asp complex in tRNA moved state,the Asp-N-tRNA Asp was mixed with ErdS variant at a molar ratio of 0.2:1 and incubatedat room temperature for 1 h. The sample was added to a previously glow discharged 300 mesh Au R1.2/1.3 grid (Quantifoil) for cryo-EM observation and rapidly frozen. Cryo-EM data were collected using a Titan Krios G3i microscope (Thermo Fisher Scientific), running at 300 kV and equipped with a Gatan Quantum-LS Energy Filter (GIF) and a Gatan K3 Summit direct electron detector in the electron counting mode. Micrographs were recorded at a nominal magnification of 105,000×, corresponding to a calibrated pixel size of 0.83Å, with a total dose of 51 electrons per Å 2 . The data were automatically collected by the image shift method using the EPU software (Thermo Fisher Scientific), with a defocus range of −1.6 to −0.4 μm, and 7,809 movies were obtained and processed using cryoSPARC v4. Initial particle picks were used as templates for repicking using Topaz, yielding 3,038,832 particles from all micrographs, and extracted. These particles were subjected to 2D classification, and the selected 354,720 particles were combined for multiple rounds of three-dimensional classification (ab initio model generation and 3D refinement). The best class containing 34,200 particles was refined using non-uniform refinement after CTF refinement, yielding a map at 3.65 Å resolution. Model building, validation and superimpositions For atomic model building, initialErdS models were generated by AlphaFold2 16 . The initial tRNAAsp was derived from the crystal structure of yeast tRNA Asp (PDB 1ASY)17. The models were manually docked into cryo-EM map and modeled in Coot andrefined using phenix.real_space_refine 17,18 . The structure validation was performed using MolProbity in the PHENIX package 19 . The statistics of the 3D reconstruction and model refinement are summarized in Extended Data Table 1. The cryo-EM density map figures and molecular graphics were generated using UCSF ChimeraX 20,21 . Models were superimposed using the match maker in ChimeraX. Ergosterol was positioned and relaxed by Sphere Refine on COOT. Fungal strains and growth media All strains and derivatives used in this study are listed in Table S1 . S. cerevisiae strains used for plasmid shuffling are also presented in Table S2 . S. cerevisiae strains BY4742 ( MATα his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0 ) were used to express WT ErdS and mutants from pRS415 (LEU) plasmids. They were routinely grown on SC-Leu agar (MP™ Biomedicals) at 30 °C. The S. cerevisiae YAL3 ( dps1 D) shuffle strain ( MATa ura3-52 lys2-801am trp1-63 his3-200 leu2-1 ade2-450 ade3-1483 dps1 :: HIS3 ) contained a rescue plasmid (TRP) and pRS415 (LEU) plasmids carrying WT of mutant versions of ErdS. Before shuffling they were grown on SC-Leu-Trp at 30 °C, and after shuffling, when the rescue plasmid was chased, on SC-Leu agar (MP™ Biomedicals). For liquid cultures, the same media without agar were used in glass flasks, cultures incubated at 30 °C under shaking (200 rpm). For Aspergillus fumigatus , the CEA17 D akuB KU80 strain ( aka “WT Ku80”) and derivatives were used A. fumigatus was maintained as follows: fresh conidia were spread on Malt agar (Thermo Scientific) plates or slants and incubated in the dark at 37 °C until it produced enough mature conidia. For phenotypic characterization, strains were grown on Malt agar or on Minimal Medium (MM) Agar plates (For 1 L, MM contained glucose (2 % w/v) or xylose (2 % w/v), ammonium tartrate dibasic (0.92 g, 5 mM, i.e. , 10 mM ammonium), salts (10 mL of a 50 X solution containing KCl 26 g/L, MgSO 4 7H2O 26 g/L, KH 2 PO 4 76 g/L) and trace elements (0.5 mL of a 1000 X solution containing: FeSO 4 7H 2 O 1 g, Na 2 EDTA 10 g, ZnSO 4 7H 2 O 4.4 g, H 3 BO 3 2.2 g, MnCl 2 4H 2 O 1 g, CoCl 2 6H 2 O 0.32 g, CuSO 4 5H 2 O 0.32 g and Na 2 MoO 4 0.8 g for 200 mL adjusted at pH 6.5 ; 1.5 % (w/v) agar was added). For liquid cultures, Afm was grown in liquid MM with 1 % (w/v) glucose. Liquid cultures were performed in glass flasks at 37 °C ( Afm ) under shaking (200 rpm) for 24 h ( Afm , Sce ) or 48 h ( Ncr ). For Neurospora crassa , the 74-OR23-1VA WT (Fungal Genetic Stock Center, strain FGSC#2489) and D erdH (FGSC#20235) strains were employed. N. crassa was grown on nutrient rich plates (Glucose 4 % w/v, Peptone 1 % w/v and Yeast extract 2 % w/v), or in MM liquid medium at 30 °C. Magnaporthe oryzae strains (isolate Guy11) were routinely grown, maintained and stored as previously described 22 . Construction of A. fumigatus strains Construction of the Afm D erdS and D erdS ::P Xyl - erdS strains was described in 23 . The WT::P Xyl - erdS strain was constructed using the same integration cassette as that used for the D erdS ::P Xyl - erdS strain. It was designed from the six- P xyl -b rec - trpC - hygB - six resistance cassette (with the b-recombinase six sequences) 24 surrounded by the 5’ and 3’ 1000-bp flanking regions of the erdS gene.The erdS gene was inserted in place of the b-recombinase gene between the P Xyl promoter and TrpC terminator 23 .Transformations of A. fumigatus CEA17 D akuB KU80 was performed by electroporation of swollen conidia essentially as described 25 and we used 10 μg of linearized ( Sma I-digested) plasmid containing the replacement cassette. From the primary hygromycin-resistant colonies, conidia were isolated on individual Malt agar plates containing 150 μg/mL hygromycin B. After 3 days, conidia from single isolated colonies were scrapped and transferred on Malt agar slants containing 150 μg/mL hygromycin B and incubated 5 days at 37 single iso DNA was extracted 26 from mycelium cultures ofmutants and replacement of the Afm-erdS gene was confirmed by PCR (Primers FF#684+NY#015 and NY#016+FF#685). Construction of Afm strains expressing ErdS-eGFP and ErdH-mCherry. With the E-Zyvec company (now Polyplus), we constructed plasmids containing the erdH—erdS locus found in Afm that we modified by fusion codon-optimized ORFs of eGFP and mCherry, respectiverly at the 3’-end of erdS and erdH . Both genes were thus under the control of their own natural promoter (P erd ). This construct was inserted before the six- P xyl -β rec - trpC - hygB - six cassette. The 5’- and 3’-flanking regions of the erdH—erdS locus were added, to obtain the 5’-flank_ erdH-mCherry—erdS-eGFP-six- P xyl -β rec - trpC - hygB - six_ 3’_flankcassette, surrounded by SmaI restriction sites. The resulting plasmid was digested using SmaI and transformed in the A. fumigatus CEA17 D akuB KU80 (WT Ku80) strain by electroporation, as described. Hygromycin-resistant colonies were selected on MM medium (pH 6.8) containing 150 µg/mL hygromycin B and purified through two successive re-streaks on Malt agar containing 150 µg/mL hygromycin. Strains that presented red and green fluorescence were then grown on MM containing 2 % (w/v) xylose to excise the six- P xyl -β rec - trpC - hygB - six cassette, and to ensure that only the 5’-flank_ erdH-mCherry—erdS-eGFP-_ 3’_flank construct remained. Two independent clones were selected for epifluorescence observations. Strains were verified by PCR. Construction of M. oryzae strains (CrispR) The erdS (MGG_13783) and erdH (MGG_04680) genomic sequences were retrieved from EnsemblFungi(https://fungi.ensembl.org/). erdS and erdH were deleted using the RNP-mediated CrispR editing strategy as previouslyadapted in M. oryzae 27 . To generate the donor DNAs, the BAR (glufosinate-ammonium resistance) cassette was amplified from the pCB1530 plasmid (FGSC) using approx. 100 bplong primers leading to amplicons containing the BAR cassette flanked by erdS or erdH homologous arms. The sgRNA were obtained by in vitro transcription using the EnGen® sgRNA Synthesis Kit (Neb #3322) following the manufacturer’s instructions. All primers used to generate and verify the strains are listed in Table S3 . Plasmids construction The Afm erdS open reading frame ( AFUA_1g02570 ) was synthesized (Genscript®) withcodon optimization for expression in Sce , amplified by PCR and cloned in a pRS415 (LEU) yeast expression vector as described in 23 . The p415- Aor-erdS plasmid containing the erdS gene from Aor was constructed previously 23 . All ErdS mutants were obtained from these pRS415 plasmids through mutagenesis. Briefly, for site-directed mutagenesis, we proceeded as follows. A pair of overlapping primers containing the desired mutation were used ( Table S3 ). The “reverse” primer was used together with a “forward” primer matching the ampicilline resistance gene of the plasmid (FF#060) to amplify by PCR a first half of the pRS415- erdS plasmid, containing the desired mutation. The “forward” primer was used with the “reverse” primer (FF#061) matching the ampicilline resistance gene of the plasmid to amplify the second half of the plasmid, with the desired region mutated. Then, 1 µL of each PCR reaction were used in a Gibson Assembly reaction mixture to reunite the two plasmid halves. Then, 8 µL of the reaction mixture was used to transform XL-1 Blue chimiocompetent E. coli cellsto select plasmids in which the ampicilline resistance gene was properly reconstituted. The same strategy was used to obtain the erdS D(1-90) mutant of Aor erdS from the pRS415- Aor erdS plasmid. The presence of the desired mutations in erdS was verified and validated by sequencing. Plasmid-shuffling complementation assays in S. cerevisiae Plasmid shuffling experiments were conducted in the YAL3D dps1 Sce strain 28 rescued with awild-type DPS1 gene copy cloned in an URA3 -bearing plasmid. All Afm or AorerdS constructsto be tested were cloned in pRS415 (LEU) plasmids, transformed in the D dps1 strain, and shuffledessentially as described 28,29 . Preparation of spores/conidia from A. fumigatus and N. crassa Spores from 7 days-old Malt agar slants or plates were resuspended by addition of 5 mL sterile Tween 20-H 2 O (0.05 % v/v) and vortexing, then the conidia were filtered with Cell Strainer filters (EASY strainer TM Greiner Bio-One), and the concentration was determined with a LUNA-FL cell counter (ref.59506, Dutscher). Conidia were stored in Tween 20-H 2 O (0.05 % v/v) at 4 °C for up to 2 week. For Ncr , conidia were harvested from 14 days-old Malt agar slants and treated similarly using 1 M sterile sorbitol.For M. oryzae , spores were harvested from 7-9 days old cultures grown on CM agar plates. 4 mL were poured on the Petri dish, and spores were released by scratching the mycelia surface using a sterile disposable plastic spreader. The spore solution was then filtered through Miracloth, centrifuged at 3000 x g for 5 min and finally resuspended in 1 mL of water. Cells were counted using a Bright-Line™ Hemacytometer (Merck #Z359629). Phenotypic characterization of A. fumigatus strains For phenotypic characterizations, 10 6 Afm conidia in 5 µL were point-inoculated on squared agar plates from suspensions prepared as indicated below to obtain individual round-shaped colonies. Each plate was inoculated with the 4 Afm derivative (WT Ku80, D erdS , D erdS ::P Xyl - erdS and WT::P Xyl - erdS ), as to ensure proper comparison between strains. Colony diameter was measured at indicated time points and colonies were pictured using a cell phone or using an AxioVision magnifier. To produce mycelia lawns, 50 µL of a conidia suspension at 10 7 conidia/mL were spread on MM agar medium-containing wells of 12-well plates. Each plate featured 3 replicates of the 4 strains tested. Lawns were pictured using an AxioVision magnifier. All plates were incubated at 37 °C in the dark and documented at the indicated time points. Liquid cultures and mycelia harvesting Liquid cultures to produce mycelia for total lipids extraction were inoculated with 10 7 conidia/mL in 50 mL liquid MMG (MM + 1 % (w/v) glucose), incubated for 24 h at 37 °C ( Afm ) or 48 h at 30 °C( Ncr ) in the dark under agitation (220 rpm). To test the overproduction of Erg-Asp, the 4 Afm strains were grown in liquid MM + 2 % (w/v) glucose (MMG) or MM + 2 % (w/v) xylose (MMX). Experiments were also conducted with cultures in the presence of various ratios of glucose and xylose. Mycelia were then filtrated through two layers of gauze, rinsed twice with 50 mL sterile H 2 O, and squeezed to eliminate excess water. Mycelia were directly used to extract total lipids. Germination assay To investigate the role of Erg-Asp in germination process, conidia suspension (1x10 6 conidia /mL) from WT Ku80 and D erdS strains were prepared. Germination assays were performed in 1 mL MM medium in Eppendorf tubes, incubated at 37 °C with agitation at 200 rpm. Microscopic observation began 3 hours after incubation, then continued hourly. At each time point, samples were collected and centrifuged at maximum speed for 10 min at 4°C. Supernatants were removed, and pellets were fixed in cold 4% paraformaldehyde (4% PFA) for 15 min at room temperature. After a second centrifugation (10 min, max speed, 4°C) and removal of the fixative, conidia were resuspended in 1mL, sterile 1xPBS (phosphate buffered saline) and stored à 4 °C until microscopy observations. Bright field images were captured hourly using a Zeiss Axiovert 200 epifluorescence microscope with a 65x objective. Germination was manually assessed by examining several fields of view for each strain and each time point. Conidia were outlined manually, totaling approximately 100 fungal cells per strain per time point. Observations were recorded hourly 3 to 8 hours post-incubation. Germination percentages at the germ tube stage were compared between WT Ku80 and Δ erdS strains over time using a two-way ANOVA statistical test performed with GraphPad Prism. Multiple comparisons were conducted using the Sidak test, with a significance threshold set at 0.05. Nanolive microscopy Cell Culture. Murine macrophage IC-21 cells were cultured under standard conditions at 37 °C with 5 % CO 2 . They were maintained in complete growth medium (RPMI+FBS+PS) and seeded for two different experiments in 35 mm ibidi low border dishes. On July 18, 2023, five dishes were seeded with 60,000 cells each, while on July 24, 2023, two additional dishes were seeded with 80,000 cells each. Infection Preparation and Conditions: For the first experiment (WT Ku80 conidia), one dish was used to assess cell viability following a 30-minute incubation at 4 °C, a condition necessary for synchronizing phagocytosis. A second dish was used for cell counting to calculate the appropriate number of spores needed to reach the desired multiplicity of infection (MOI). The remaining three dishes were used for infection with WT Ku80 spores: one dish served as a negative control, one was infected at MOI 5, and the third at MOI 10. To achieve this, the stock solution (2.36 × 10⁹ spores in 4 mL) was first pre-diluted 1:200 (3 µL stock + 597 µL medium) to obtain 1.77 × 10⁶ spores in 600 µL. For an MOI of 10, 237 µL of this pre-dilution (corresponding to 700,000 spores) was added to 70,000 IC-21 cells in a final volume of 800 µL. For an MOI of 5, 119 µL (350,000 spores) was used under the same conditions.After 24 h of incubation, spores were added to the respective dishes. To synchronize phagocytosis, all infected dishes were placed at 4 °C for 30 min, followed by a medium change to remove unbound spores.In the second experiment ( ∆erdS spores) realized on July 24, 2023, IC-21 cells were infected with ∆erdS spores at MOI 5 and MOI 10. In this case, the stock solution (3.28 × 10⁹ spores in 4 mL) was also pre-diluted 1:200 (3 µL stock + 597 µL medium) to obtain 2.46 × 10⁶ spores in 600 µL. For an MOI of 10, 196 µL of this pre-dilution (corresponding to 800,000 spores) was added to 80,000 IC-21 cells per dish in a final volume of 800 µL. For an MOI of 5, 98 µL (400,000 spores) was used under the same conditions. The spore solution was taken out of the fridge for 15 min before dilution to reach room temperature and the cells were taken out of the incubator 5 min before adding the spores. As in the previous experiment, cells were incubated with spores at 4 °C for 30 min, then washed by changing the medium to eliminate unbound spores.All Afm spore preparations were performed one by one with complete disinfection in between to avoid any risk of cross-contamination between spore types. Image Acquisition: Following spore addition and washing, live-cell imaging was performed using a CX96-Focus (Nanolive SA, Tolochenaz, Switzerland) microscope. The microscope is equipped for long-term live cell imaging: temperature, humidity, and gas composition. The incubator chamber (Okolab) keeps the sample at 37 °C, is closed by a heating glass lid to prevent condensation and is connected to a gas mixer (2GF-Mixer, Okolab) to maintain 5% of CO 2 . The humidity module ensures a 90% relative humidity within the chamber. The first experiment was done with a 3×3 grid scan with one image every 2min9s, while the second used a 4×4 grid scan with one image every 2min21s. Fluorescence microscopy of A. fumigatus Conidia from two independent Afm strains carrying the erdH-mCherry—erdS-eGFP locus were used to inoculate liquid YG medium (1 % Yeast extract, 2 % glucose) (10 5 conidia/mL). Development of the fungus was followed at the indicated time points. At each time point, 1 mL of culture was aliquoted, conidia or mycelia centrifugated and washed with phosphate buffer saline (PBS), resuspended in PBS and 10 µL of resuspended strains were used to prepare slides. Epifluorescence imageswere taken with an AXIO Observer d1 (Carl Zeiss) epifluorescence microscope using a 100 × plan apochromatic objective (Carl Zeiss) and processed with the Image J software. Total lipids extraction Overnight Sce cultures were diluted to an OD 600 = 0.1, grown (220 rpm) until the OD 600 was ~1 and cells were harvested by centrifugation at 5000× g for 15 min at 4°C. Afm or Ncr conidia were harvested as described and inoculated in glass flasks (50 mL) in the MMG medium at a concentration of 10 6 conidia/mL, shaken at 37 °C during 24 h ( Afm ) or 30 °C for 48 h ( Ncr ). Mycelia were collected as described above. For total lipid extraction, 50 OD 600 a pellet of PBS-washed Sce cells were resuspended in 0.5 mL of 120 mM Na-Acetate, pH 4.5. Then, 3.75 volumes of CHCl 3 :CH 3 OH (2:1) and 1 mL of glass beads (ø 0.25-0.5 mm, Roth) were added and cells disrupted through mechanical lysis using a FastPrep Instrument (MP™ Biomedicals, Serial N° 10020698) at 1 min 5.5 m/s repeated 6 times with cooling on ice between each cycle. Cell lysates were incubated 3 h on a rotating wheel at 4°C. Then, 1.25 volumes of CHCl 3 and 1.25 volumes of 120 mM Na-Acetate pH 4.5 were added and the samples vortexed 1 min. Phases were separated by centrifugation (9000× g ; 30 min; 4°C) and the lower organic phase containing lipids was transferred into a clean glass tube and dried under vacuum (SpeedVac vacuum concentrator). Drying was finalized under an argon flow. Lipids were stored at -20°C or resuspended in 50-100 µL of CHCl 3 :CH 3 OH (1:1, v:v) for analysis on TLC. In the case of Afm and Ncr strains, mechanical cell disruption was performed as follows: 2g of fresh mycelia, dried on paper towel, were ground in a mortar with a pestle in the presence of liquid nitrogen and the resulting fine powder was resuspended in 1 mL (1 vol.) of 120 mM Na-Acetate pH 4.5 and treated as described above. For Afm conidia total lipids, 7-day old mycelial lawns grown on on malt agar plates were flooded with 10 mL of sterile H 2 O/0.05 % Tween-20 and conidia scrapped off the plate, filtered through a 40 µm cell strainer (ref. 0999225, Grosseron) and centrifuged 20 min, 8000x g at 4 °C. The supernatants were discarded and spores treated as described for Sce cells. Lipid analysis by Thin-Layer Chromatography (TLC) TLC plates (Silica gel 60 aluminum foils, Sigma-Aldrich, 10 x 10 cm) were used. Lipids in the CHCl 3 :CH 3 OH (1:1, v:v) solvent were spotted on TLC, typically, 10-20 µL of total lipids or 25 µL of radiolabeled lipids extracted from in vitro reactions (see below). TLCs were developed with the CHCl 3 :CH 3 OH:H 2 O mobile phase (130:50:8) for 10 min, and air-dried. TLCs were stained with a sulfuric acid/MnCl 2 solution (concentrated sulfuric acid 9 mL, MnCl 2 .4H 2 O 0.8 g, CH 3 OH 120 mL, H 2 O 120 mL) or with ninhydrin (Sigma-Aldrich, 0.4 % w/v in EtOH 100 %, v/v) and heated at 100 °C, 15 min. Plates were imaged under white light or at 254 nm. Radiolabeled lipid species were revealed by exposing TLC plates onto a Fuji Imaging Plate and analyzed with a Typhoon TRIO, Variable mode imager (GE Healthcare). In Vitro lipid aminoacylation assay Reactions were performed in the following mix: 100 mM Na-HepespH 7.2 buffer containing 30 mM KCl, 12 mM MgCl 2 , 10 mM ATP, 0.1 mg/mL bovine serum albumin, pure yeast tRNA Asp (10 μM) (46), 10 µM [U- 14 C]-Asp (280 cpm/pmol, Perkin-Elmer, NEC268E050UC) in a final volume of 50 μL.To test the transfer of [ 14 C]-Asp onto lipids, total lipids or pure sterols were added to a finalconcentration of 2 mg/mL, and commercial pure sterols were added to a final concentration of 0.5 mg/mL.Purified ErdSDN86 was added to initiate the reaction before a 45-min-long incubation at 30 °C. After incubation, reactions were stopped with addition of 500 μL ofCHCl3:CH 3 OH:120 mM Na-acetate pH 4.5 (130:50:8, v/v/v) and vortexing. Then, 130 μL of CHCl 3 and 130 μL of Na-acetate 120 mM pH 4.5 were successively added, and the mixture was vortexed and centrifuged for 1 min at 5,000 × g (RT). The lower organic phase was recovered and dried under vacuum. Reaction products were then dissolved in a CHCl 3 :CH 3 OH (1:1, v:v) mixture, spotted on TLC plates and separated with the CHCl 3 :CH 3 OH:H 2 O mobile phase. TLC plates were exposed onto an imaging plate (Fuji Imaging plate) for at least 2 h. Radioactivity was detected using a Typhoon TRIO variable Mode Imager (GE Healthcare). Quantification of radioactivespots or Erg-Asp and phosphoethanolamine (PE) bands was performed using the ImageJ software (number of pixels). Protein extraction and Western blots Yeast total proteins from Sce were extracted using 1 OD600nm of cells that were resuspended and incubated 10 min in 500 μL of pre-cooled NaOH 0.185 N, then precipitated by adding 50 μL of Trichloroacetic acid (TCA) 100 % and incubated 10 min on ice. Finally, the samples were centrifuged at 13, 000 x g for 15 min and the resulting pellets were resuspended in 100 μL of Laemmli Sample Buffer. Then, 8 μL of each sample was resolved on 10 % SDS-PAGE gels. Samples were separated by using a BioRad Mini-PROTEAN electrophoresis apparatus.For western blotting, proteins were transferred onto PVDF membranes that were blocked in 5% (w/v) skimmed milk in TBS-Tween (TBS 1X, Tween-20 0.3 % (v/v)) for 1 h at RT. Primaryantibodies (polyclonal anti-DUF2156, Covalab, France, anti-PGK, dilution 1/10,000) were incubated overnight at4 were incubated overnight a times with TBS-Tween. Membranes were then incubated for 1 hwith HRP-conjugated secondary antibodies (Goat anti-rabbit for anti-DUF2156 and Goat antimousefor anti-PGK) at RT. Revelation was performed with the BioRad clarity western ECLKit and monitored in a BioRadChemiDocTouch®apparatus. ErdS sequence alignments The sequence of Afm ErdS was used as the probe to search homologs using BLAST with the default parameters. Then, 98 homologous sequences were selected across Dikarya and aligned using MUSCLE 30 , and the resulting alignment was used to produce a sequence logo at WebLogo 3 (https://weblogo.threeplusone.com/create.cgi). Data availability The atomic models have been deposited in the PDB under the accession codes #### (ErdS tetramer), #### (ErdS/tRNA dimer on tRNA located AspRS) and #### (ErdS/tRNA dimer on tRNA located ATT). The cryo-EM density map has been deposited in the Electron Microscopy Data Bank under the accession codes EMD-##### (ErdS tetramer), EMD-##### (ErdS/tRNA dimer on tRNA located AspRS) and EMD-##### (ErdS/tRNA dimer on tRNA located ATT). All data are available in this Article or its Supplementary Information. Method References Fechter, P., Rudinger, J., Giegé, R. &Théobald-Dietrich, A. Ribozyme processed tRNA transcripts with unfriendly internal promoter for T7 RNA polymerase: Production and activity. FEBS Lett 436 , 99–103 (1998). Gamper, H. & Hou, Y.-M. tRNA 3′-amino-tailing for stable amino acid attachment. RNA 24 , 1878–1885 (2018). Gamper, H. & Hou, Y. M. A label-free assay for aminoacylation of tRNA. Genes (Basel) 11 , 1–15 (2020). Murakami, H., Ohta, A., Ashigai, H. & Suga, H. A highly flexible tRNA acylation method for non-natural polypeptide synthesis. Nat Methods 3 , 357–359 (2006). Katoh, T. & Suga, H. Flexizyme-catalyzed synthesis of 3′-aminoacyl-NH-tRNAs. Nucleic Acids Res 47 , (2019). Mastronarde, D. N. Automated electron microscope tomography using robust prediction of specimen movements. J Struct Biol 152 , 36–51 (2005). Zheng, S. Q. et al. MotionCor2: Anisotropic correction of beam-induced motion for improved cryo-electron microscopy. Nature Methods vol. 14 331–332 Preprint at https://doi.org/10.1038/nmeth.4193 (2017). Scheres, S. H. W. RELION: Implementation of a Bayesian approach to cryo-EM structure determination. J Struct Biol 180 , 519–530 (2012). Zivanov, J. et al. New tools for automated high-resolution cryo-EM structure determination in RELION-3. Elife 7 , (2018). Rohou, A. &Grigorieff, N. CTFFIND4: Fast and accurate defocus estimation from electron micrographs. J Struct Biol 192 , 216–221 (2015). Zivanov, J., Nakane, T. &Scheres, S. H. W. A Bayesian approach to beam-induced motion correction in cryo-EM single-particle analysis. IUCrJ 6 , 5–17 (2019). Rosenthal, P. B. & Henderson, R. Optimal determination of particle orientation, absolute hand, and contrast loss in single-particle electron cryomicroscopy. J Mol Biol 333 , 721–745 (2003). Punjani, A., Rubinstein, J. L., Fleet, D. J. & Brubaker, M. A. CryoSPARC: Algorithms for rapid unsupervised cryo-EM structure determination. Nat Methods 14 , 290–296 (2017). Bepler, T. et al. Positive-unlabeled convolutional neural networks for particle picking in cryo-electron micrographs. Nat Methods 16 , 1153–1160 (2019). Punjani, A., Zhang, H. & Fleet, D. J. Non-uniform refinement: adaptive regularization improves single-particle cryo-EM reconstruction. Nat Methods 17 , 1214–1221 (2020). Jumper, J. et al. Highly accurate protein structure prediction with AlphaFold. Nature 596 , 583–589 (2021). Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot . Acta Crystallogr D Biol Crystallogr 66 , 486–501 (2010). Nicholls, R. A., Tykac, M., Kovalevskiy, O. &Murshudov, G. N. Current approaches for the fitting and refinement of atomic models into cryo-EM maps using CCP-EM . Acta Crystallogr D Struct Biol 74 , 492–505 (2018). Chen, V. B. et al.MolProbity : all-atom structure validation for macromolecular crystallography. Acta Crystallogr D Biol Crystallogr 66 , 12–21 (2010). Pettersen, E. F. et al. UCSF ChimeraX: Structure visualization for researchers, educators, and developers. Protein Science 30 , 70–82 (2021). Goddard, T. D. et al. UCSF ChimeraX: Meeting modern challenges in visualization and analysis. Protein Science 27 , 14–25 (2018). Talbot, N. J., Ebbole, D. J. & Hamer, J. E. Identification and characterization of MPG1, a gene involved in pathogenicity from the rice blast fungus Magnaporthe grisea. Plant Cell 5 , 1575–1590 (1993). Yakobov, N. et al. RNA-dependent sterol aspartylation in fungi. Proc. Natl Acad. Sci. USA 117 , 14948–14957 (2020). Hartmann, T. et al. Validation of a Self-Excising Marker in the Human Pathogen Aspergillus fumigatus by Employing the β-Rec/ six Site-Specific Recombination System. Appl Environ Microbiol 76 , 6313–6317 (2010). Lambou, K., Lamarre, C., Beau, R., Dufour, N. &Latge, J. Functional analysis of the superoxide dismutase family in Aspergillus fumigatus . Mol Microbiol 75 , 910–923 (2010). Müller, F.-M. C. et al. Rapid Extraction of Genomic DNA from Medically Important Yeasts and Filamentous Fungi by High-Speed Cell Disruption. J Clin Microbiol 36 , 1625–1629 (1998). Foster, A. J. et al. CRISPR-Cas9 ribonucleoprotein-mediated co-editing and counterselection in the rice blast fungus. Sci Rep 8 , (2018). Ador, L. et al. Active site mapping of yeast aspartyl-tRNA synthetase by in vivo selection of enzyme mutations lethal for cell growth. J Mol Biol 288 , 231–242 (1999). Sikorski, R. S. & Boeke, J. D. [20] In vitro mutagenesis and plasmid shuffling: From cloned gene to mutant yeast. in 302–318 (1991). doi:10.1016/0076-6879(91)94023-6. Additional Declarations There is NO Competing Interest. Supplementary Files TableS1.pdf Table S1 TableS2.png Table S2 TableS3.xlsx Table S3 ErdSSupplementary.pdf Supplementary Information and Figures 1-11 Movie1.mp4 Supplementary Movie 1 Movie2.mp4 Supplementary Movie 2 Movie3.mp4 Supplementary Movie 3 Cite Share Download PDF Status: Under Review Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7291846","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":503269409,"identity":"078a60cf-046a-4cca-8062-58209abb6f0e","order_by":0,"name":"Osamu 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13:46:23","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7291846/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7291846/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":89624495,"identity":"985b98f8-735f-4d47-b4f8-1af024f7622e","added_by":"auto","created_at":"2025-08-22 05:31:44","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1303964,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eErg-Asp synthesis in the physiology of fungi. A. \u003c/strong\u003eErdS is composed of an aspartyl-tRNA synthetase (AspRS) domain (blue) and an ATT domain (orange). The first binds tRNA\u003csup\u003eAsp\u003c/sup\u003e (1), L-Asp, and ATP and activates Asp into Asp~AMP, and the activated Asp is transferred onto the 3’-OH group of tRNA\u003csup\u003eAsp\u003c/sup\u003e, yielding Asp-tRNA\u003csup\u003eAsp\u003c/sup\u003e (2). The acceptor arm of aspartylated tRNA\u003csup\u003eAsp\u003c/sup\u003e is believed to translocate towards the ATT domain (3), where the Asp is transferred onto the 3β-OH group of Erg, producing Erg-Asp (4), whose structure is represented on the right side. Then, tRNA\u003csup\u003eAsp\u003c/sup\u003e can be released (5). ErdH can hydrolyze Erg-Asp back to Erg and Asp (6). \u003cstrong\u003eB. \u003c/strong\u003eGrowth of colonies of the indicated \u003cem\u003eAfm\u003c/em\u003e strains on malt agar supplemented with Glc (repressing condition) or Xyl (inducing condition). Pictures present colony morphologies after 48 h. Conidiation (green) areas were measured (fraction of total colony area) in each strain, in addition to colony diameters (see Fig. S1C). \u003cstrong\u003eC. \u003c/strong\u003eGrowth of colonies of the indicated \u003cem\u003eAfm\u003c/em\u003e strains on MM supplemented with Glc or Xyl. Colonies were imaged using a magnifier to visualize the fluffy phenotype (arrowheads), which is qualitatively described. \u003cstrong\u003eD. \u003c/strong\u003eGrowth of mycelia lawns of the indicated \u003cem\u003eAfm\u003c/em\u003e strains on MM supplemented with Glc or Xyl in 12-well plates, after 20 or 48 h. Lawns were imaged with a magnifier at each time point to visualize the presence or absence of conidiophores. \u003cstrong\u003eE. \u003c/strong\u003e\u003cem\u003eLeft. \u003c/em\u003eTLC analyses of total lipids from WT \u003cem\u003eMor\u003c/em\u003e and clones of \u003cem\u003eerdH\u003c/em\u003e and \u003cem\u003eerdS\u003c/em\u003e mutant strains. \u003cem\u003eRight.\u003c/em\u003e TLC showing separation of total lipids from the WT and \u003cem\u003eerdS\u003c/em\u003e strains of \u003cem\u003eAfm\u003c/em\u003e and the WT and \u003cem\u003eerdH\u003c/em\u003e strains of \u003cem\u003eNcr\u003c/em\u003e. In the latter, as in \u003cem\u003eMor\u003c/em\u003e, Erg-Asp is undetectable unless \u003cem\u003eerdH\u003c/em\u003e is deleted. Asterisks (*) indicate Erg-Asp bands. \u003cstrong\u003eF. \u003c/strong\u003eColony morphologies of WT, \u003cem\u003eerdS\u003c/em\u003e and \u003cem\u003eerdH\u003c/em\u003e strains of \u003cem\u003eMor\u003c/em\u003e on plates. Plates were illuminated from below (first row), or from above (second row). The third row presents colonies imaged from below. Bars represent the number of conidia counted per plate after 9 days of culture.\u003c/p\u003e","description":"","filename":"ErdSFigs1.png","url":"https://assets-eu.researchsquare.com/files/rs-7291846/v1/e20ca8ffb0c6684e39b661f1.png"},{"id":89624506,"identity":"138873c0-d34d-491a-8c11-dbe7d53d6ef7","added_by":"auto","created_at":"2025-08-22 05:31:44","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1053374,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of the \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eerdS\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e mutation on germination and stress tolerance, and dynamic localizations of ErdS and ErdH in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eAfm\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e. A. \u003c/strong\u003eGermination kinetics of WT, \u003cem\u003eerdS,\u003c/em\u003e and \u003cem\u003eerdS\u003c/em\u003e::P\u003cem\u003eXyl\u003c/em\u003e-\u003cem\u003eerdS\u003c/em\u003e strains. The graph presents the percentages of swollen conidia (isotropic growth) observed between 3 and 9 hours. \u003cstrong\u003eB. \u003c/strong\u003eKaplan-Meier curves showing the distinct germination dynamics between strains in under MOI conditions. Confidence intervals (95%) illustrate the differences in variability and spore behavior across conditions. \u003cstrong\u003eC. \u003c/strong\u003eFluorescence microscopy of the \u003cem\u003eAfm\u003c/em\u003e strain carrying the \u003cem\u003eerdH-mCherry—erdS-eGFP \u003c/em\u003ecluster at four stages of fungal development (illustrated on the right side and Fig. S1B). \u003cstrong\u003eD. \u003c/strong\u003eCongo Red (CR) sensitivity tests on MM + glucose plates supplemented with increasing concentrations of CR. Three strains (indicated on the left) were tested in three replicates (\u003cem\u003eRep. 1 \u003c/em\u003eto \u003cem\u003e3\u003c/em\u003e). Images were taken after 48 hours of growth. \u003cstrong\u003eE. \u003c/strong\u003eStress sensitivity tests conducted on the WT and \u003cem\u003eerdS\u003c/em\u003e strains under the indicated stress conditions.\u003c/p\u003e","description":"","filename":"ErdSFigs2.png","url":"https://assets-eu.researchsquare.com/files/rs-7291846/v1/d10762cc688ea7217fc813d3.png"},{"id":89624499,"identity":"1d8afea3-eee0-48f7-820b-efbd98d626ed","added_by":"auto","created_at":"2025-08-22 05:31:44","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1731365,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eStructure and mutational characterization of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eAfm\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e ErdS and its sterol specificity. A. \u003c/strong\u003eStructure of the ErdS dimer. For comparison, the architecture of ErdS is represented with the position of each domain numbered according to that for \u003cem\u003eAfm\u003c/em\u003e. AspRSs are colored blue (AspRS 1) and light blue (AspRS 2), and ATTs are orange (ATT 1) and light orange (ATT 2). NTD: N-terminal domain; CD: catalytic domain of AspRS; ABD: anticodon-binding domain; α\u003csup\u003e(+)\u003c/sup\u003e: positively charged alpha helix. \u003cstrong\u003eB.\u003c/strong\u003e Structure of the ATT domain: GNAT I subdomain is dark grey, GNAT II is orange, and the α\u003csup\u003e(+)\u003c/sup\u003e helix is purple. The sequence of this helix in \u003cem\u003eAfm\u003c/em\u003e is indicated. \u003cstrong\u003eC. \u003c/strong\u003eStructural comparison between the ATT domain of ErdS (orange) and the GNATⅡ domain of FemX (white) with its substrates (light gray) (PDB: 4II9). Ergosterol binding pocket of the ATT domain. The tRNA CCA analog was superimposed. Important residues are represented with a color code reflecting the effects of mutations of these residues on the activity (see \u003cstrong\u003eD\u003c/strong\u003e). \u003cstrong\u003eD. \u003c/strong\u003eMutational analysis of ErdS. ErdS activity was determined (bars) for each mutant (indicated above) in total lipids of \u003cem\u003eSce\u003c/em\u003e separated by TLC (second row), by calculating the ratio of the Erg-Asp signal measured in the corresponding migration area and the signal of the phosphatidylethanolamine (PE) that migrates slightly above Erg-Asp (Erg-Asp/PE ratio (\u003cem\u003eR\u003c/em\u003e)). In the absence of Erg-Asp, phosphatidylglycerol becomes visible, which results in an \u003cem\u003eR\u003c/em\u003e ratio not equal to zero. Immunoblots show the expression of ErdS using anti-ATT antibodies and the loading control (stain-free). Residues corresponding to those mutated in Afm are shown for the \u003cem\u003eAor\u003c/em\u003e, \u003cem\u003eNcr\u003c/em\u003e, and \u003cem\u003eMor \u003c/em\u003eErdSs. \u003cstrong\u003eE. \u003c/strong\u003eOrganization of the Erg-binding domain. Erg and the CCA analog are shown. \u003cstrong\u003eF. \u003c/strong\u003eDetails of the Erg-binding site with docked Erg. Important residues are represented in a color code reflecting the effects of their mutations on the activity (as depicted in \u003cstrong\u003eC\u003c/strong\u003e). \u003cstrong\u003eG.\u003c/strong\u003e TLC showing products formed with \u003cem\u003ein vitro\u003c/em\u003e sterol aminoacylation assays. Sterols used are indicated. The control TLC shows the Cho, Pregnenolone (Preg.), and DHA bands from stock sterols, and illustrates that no visible Cho seems to contaminate Preg. or DHA. Ctrl: no sterols. \u003cstrong\u003eH.\u003c/strong\u003e Structures of the sterols used in \u003cstrong\u003eG\u003c/strong\u003e.\u003c/p\u003e","description":"","filename":"ErdSFigs3.png","url":"https://assets-eu.researchsquare.com/files/rs-7291846/v1/5b357674187703106e5139d8.png"},{"id":89624500,"identity":"6372be68-50da-4d1b-a8ba-a7cf0f87e393","added_by":"auto","created_at":"2025-08-22 05:31:44","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1307973,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe NTD maintains tRNA\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003eAsp\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e with ErdS. A. \u003c/strong\u003eAlignment of the NTDs from the \u003cem\u003eAfm\u003c/em\u003e, \u003cem\u003eAor\u003c/em\u003e, \u003cem\u003eNcr\u003c/em\u003e, and \u003cem\u003eMor \u003c/em\u003eErdSs. Positively charged residues are blue, residues that interact with the anticodon stem are red, and the region involved in the interaction with the elbow region is highlighted in yellow. \u003cstrong\u003eB. \u003c/strong\u003eStructure of ErdS with the NTD and tRNA\u003csup\u003eAsp\u003c/sup\u003e. \u003cstrong\u003eC, D. \u003c/strong\u003eClose-up view of the contacts between the ErdS N-terminal region and tRNA\u003csup\u003eAsp\u003c/sup\u003e. \u003cstrong\u003eE. \u003c/strong\u003eElectrostatic potential of the NTD (blue – basic; red – acidic; white - neutral). \u003cstrong\u003eF. \u003c/strong\u003eComplementation of the \u003cem\u003eSce \u003c/em\u003eYAL3 \u003cem\u003edps1\u003c/em\u003e strain with the indicated proteins and ErdS variants. Pictures represent \u003cem\u003eSce\u003c/em\u003e colonies on plates after 3 days of growth. Lower panel: TLC profiling of total lipids from these strains to visualize Erg-Asp production (visualization under UV light). Asterisks (*) indicate Erg-Asp bands. \u003cstrong\u003eG. \u003c/strong\u003eComplementation of the \u003cem\u003eSce \u003c/em\u003eYAL3 \u003cem\u003edps1\u003c/em\u003e strain with the indicated \u003cem\u003eAor \u003c/em\u003eErdS variants. Pictures represent \u003cem\u003eSce\u003c/em\u003e colonies on plates after 3 days of growth.\u0026nbsp; Two clones of the \u003cem\u003eAor \u003c/em\u003eErdS ΔN90 were used. Arrows indicate positions of several extremely slow-growing colonies. Lower panel: TLC profiling of total lipids from these strains to visualize Erg-Asp production (visualization under UV light). Asterisks (*) indicate Erg-Asp bands.\u003c/p\u003e","description":"","filename":"ErdSFigs4.png","url":"https://assets-eu.researchsquare.com/files/rs-7291846/v1/e7bef599dfa55d81eb404cff.png"},{"id":89624497,"identity":"67ed9991-2365-420e-b3ea-d6620b33da92","added_by":"auto","created_at":"2025-08-22 05:31:44","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1313562,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eStructure of the ErdS-tRNA\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003eAsp\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e complex. A. \u003c/strong\u003eCryo-EM density map of the ErdS/tRNA tetramer with ATT domains. \u003cstrong\u003eB.\u003c/strong\u003e Cryo-EM density map of ErdS/tRNA on the tRNA acceptor stem in the moved state. The other protomer in the ErdS/tRNA model is superimposed based on the AspRS position. \u003cstrong\u003eC.\u003c/strong\u003e Superimposition of the tRNA structures when the acceptor arm is ATT-bound (purple) or AspRS-bound (gray). \u003cstrong\u003eD. \u003c/strong\u003eScheme of tRNA acceptor stem movement. The acceptor stem is translocated from AspRS to ATT in \u003cem\u003etrans\u003c/em\u003e. \u003cstrong\u003eE. \u003c/strong\u003eStructure of the ErdS/Asp-N-tRNA on the tRNA acceptor stem in the moved state. ATT2 is superimposed based on the AspRS position.\u003c/p\u003e","description":"","filename":"ErdSFigs5.png","url":"https://assets-eu.researchsquare.com/files/rs-7291846/v1/2d8cdda62da10363d489f897.png"},{"id":89624494,"identity":"41e1e8e6-d823-449d-979f-63bc3a82f1aa","added_by":"auto","created_at":"2025-08-22 05:31:44","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1676234,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eProposed Erg aspartylation mechanism by ErdS. \u003c/strong\u003eScreen captures from Supplementary Movie 3, showing ATT2 (light orange) and tRNA acceptor stem movements throughout the catalytic cycle of ErdS.\u003cstrong\u003e (1) \u003c/strong\u003eDescription of the ErdS/tRNA\u003csup\u003eAsp\u003c/sup\u003e dimer complex with the tRNA\u003csub\u003e1\u003c/sub\u003e\u003csup\u003eAsp\u003c/sup\u003e (dark grey) acceptor arm in the AspRS1 (blue) active site. The anticodon of tRNA\u003csub\u003e1\u003c/sub\u003e\u003csup\u003eAsp\u003c/sup\u003e is bound to ABD1 and further stabilized by NTD1 (red) and ATT1 (orange).\u003cstrong\u003e \u003c/strong\u003eIn\u003cstrong\u003e (2) \u003c/strong\u003eand \u003cstrong\u003e(3) \u003c/strong\u003eATT2, initially bound to tRNA\u003csub\u003e2\u003c/sub\u003e\u003csup\u003eAsp\u003c/sup\u003e, rotates and approaches tRNA\u003csub\u003e1\u003c/sub\u003e\u003csup\u003eAsp\u003c/sup\u003e, with helix a\u003csup\u003e(+)\u003c/sup\u003e positioned on the acceptor arm. Then \u003cstrong\u003e(4, 5)\u003c/strong\u003e, the acceptor arm of tRNA\u003csub\u003e1\u003c/sub\u003e\u003csup\u003eAsp\u003c/sup\u003e moves from the AspRS1 active site to ATT2 and is likely constrained by the α\u003csup\u003e(+)\u003c/sup\u003e helix. NTD1 retains tRNA\u003csub\u003e1\u003c/sub\u003e\u003csup\u003eAsp\u003c/sup\u003e and limits the dissociation of Asp-tRNA\u003csub\u003e1\u003c/sub\u003e\u003csup\u003eAsp\u003c/sup\u003e. Finally \u003cstrong\u003e(6)\u003c/strong\u003e, the CCA extends into the GNAT II active site, where Asp is transferred to the 3-OH position of Erg. ATT2 therefore acts \u003cem\u003ein trans\u003c/em\u003e. Finally, the acceptor stem of tRNA\u003csub\u003e1\u003c/sub\u003e\u003csup\u003eAsp\u003c/sup\u003e can shift back into the AspRS active site to be used for another round of aspartylation, and ATT2 can fold back to its initial position. Residues W785, R789 and K916, which are important for the ErdS activity, are indicated.\u003c/p\u003e","description":"","filename":"ErdSFigs6.png","url":"https://assets-eu.researchsquare.com/files/rs-7291846/v1/3c820605593e3d9089f53bc8.png"},{"id":89626508,"identity":"7a26db7e-b70b-47cd-8079-7dcc27daf6e8","added_by":"auto","created_at":"2025-08-22 05:55:49","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":10880032,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7291846/v1/ce54d182-79aa-4fb0-8223-d66f332de4b7.pdf"},{"id":89624507,"identity":"2cc35ee4-3fd1-4790-b7b2-2dde90525728","added_by":"auto","created_at":"2025-08-22 05:31:45","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":224707,"visible":true,"origin":"","legend":"Table S1","description":"","filename":"TableS1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7291846/v1/76df162b010ab19228e10118.pdf"},{"id":89625299,"identity":"ac6a556a-48af-43d3-b717-cdd85cdbdd68","added_by":"auto","created_at":"2025-08-22 05:39:44","extension":"png","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":4179042,"visible":true,"origin":"","legend":"Table S2","description":"","filename":"TableS2.png","url":"https://assets-eu.researchsquare.com/files/rs-7291846/v1/ac7d51eb9a97e25acdb418e6.png"},{"id":89624502,"identity":"455f3010-490c-4acf-9263-9830f986521d","added_by":"auto","created_at":"2025-08-22 05:31:44","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":19863,"visible":true,"origin":"","legend":"Table S3","description":"","filename":"TableS3.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7291846/v1/d150eeae026872115dc5500f.xlsx"},{"id":89625303,"identity":"e60de3f0-944c-42cc-80ec-daa86f6f9075","added_by":"auto","created_at":"2025-08-22 05:39:45","extension":"pdf","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":12027267,"visible":true,"origin":"","legend":"Supplementary Information and Figures 1-11","description":"","filename":"ErdSSupplementary.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7291846/v1/e0d87c5359f22f69d845d9ce.pdf"},{"id":89624516,"identity":"15048f26-c3c9-45f1-b2f0-39820c7e4db4","added_by":"auto","created_at":"2025-08-22 05:31:45","extension":"mp4","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":70710145,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Movie 1\u003c/p\u003e","description":"","filename":"Movie1.mp4","url":"https://assets-eu.researchsquare.com/files/rs-7291846/v1/3151be04f01f67c205156ab2.mp4"},{"id":89624517,"identity":"8fd69f5d-926b-4d12-a8c7-80c65a2be058","added_by":"auto","created_at":"2025-08-22 05:31:46","extension":"mp4","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":51413230,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Movie 2\u003c/p\u003e","description":"","filename":"Movie2.mp4","url":"https://assets-eu.researchsquare.com/files/rs-7291846/v1/36662a675ff548d8a8f08dd3.mp4"},{"id":89624512,"identity":"83bd7255-1ee2-41d2-92c8-82fb8659b3f5","added_by":"auto","created_at":"2025-08-22 05:31:45","extension":"mp4","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":13654079,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Movie 3\u003c/p\u003e","description":"","filename":"Movie3.mp4","url":"https://assets-eu.researchsquare.com/files/rs-7291846/v1/8fb1411ce39daedb17f0e2cc.mp4"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Structural basis for tRNA-dependent sterol aminoacylation underlying cell membrane integrity","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAminoacylated transfer RNAs (aa-tRNAs) play an essential role in delivering amino acids (aa) to translating ribosomes. Recent studies have shown that aa-tRNAs also serve as a source of activated aa in a wide range of other cellular processes unrelated to protein synthesis\u003csup\u003e\u003cspan additionalcitationids=\"CR2 CR3 CR4\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. In these alternate routes, the tRNA-esterified aa moiety is transferred onto hydroxyl- or amino- groups of diverse acceptor molecules by enzymes called aa-tRNA transferases (ATTs). One structural family of these ATTs shares a common double Gcn5-\u003cem\u003eN\u003c/em\u003e-acetyltransferase (\u003cem\u003ed\u003c/em\u003eGNAT)-like fold, and participates in a variety of antibiotic resistance and pathogenicity pathways via aminoacylations of peptidoglycan and glycerolipids in bacteria\u003csup\u003e\u003cspan additionalcitationids=\"CR7 CR8 CR9\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. In higher fungi\u0026mdash;including human and plant pathogens\u0026mdash;we recently identified two new \u003cem\u003ed\u003c/em\u003eGNAT-related ATTs, named ergosteryl-3β-\u003cem\u003eO\u003c/em\u003e-(L)-amino acid (Erg-aa) synthases (ErxS), which aa-tRNA-dependently conjugate aa to sterols\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. The first discovered was the ergosteryl-3β-\u003cem\u003eO\u003c/em\u003e-L-aspartate synthase (ErdS), which catalyzes the aspartylation of the 3β-OH group of ergosterol (Erg)\u003csup\u003e\u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e, yielding ergosteryl-3β-\u003cem\u003eO\u003c/em\u003e-L-aspartate (Erg-Asp; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Concomitantly, we found an Erg-Asp hydrolase (ErdH) that deacylates Erg-Asp back to Erg and aspartate (Asp)\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. This latter enzyme is an ergosteryl-3β-\u003cem\u003eO\u003c/em\u003e-glycine (Erg-Gly) synthase (ErgS), and only found in ascomycota.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eErdS is a bifunctional enzyme with unique structural features: the aspartyl-tRNA synthetase (AspRS) domain first activates Asp through adenylation and esterifies it to tRNA\u003csup\u003eAsp\u003c/sup\u003e, while the appended ATT domain\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan additionalcitationids=\"CR15\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e transfers the aspartyl moiety from Asp-tRNA\u003csup\u003eAsp\u003c/sup\u003e to the 3β-OH group of Erg\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. Deletion of the \u003cem\u003eerdS\u003c/em\u003e gene in \u003cem\u003eAspergillus fumigatus\u003c/em\u003e (\u003cem\u003eAfm\u003c/em\u003e) and \u003cem\u003eAspergillus oryzae\u003c/em\u003e (\u003cem\u003eAor\u003c/em\u003e) had no effect on growth, suggesting that the Asp-tRNA\u003csup\u003eAsp\u003c/sup\u003e synthesized by ErdS does not significantly contribute to normal biological activities such as protein synthesis, which utilizes a different, classical AspRS\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. This bi-functional reaction reminds us of a mechanism observed in other aa-tRNA utilizing enzymes in which, even after aspartylation, tRNA\u003csup\u003eAsp\u003c/sup\u003e remains bound to the AspRS domain of ErdS, with only the tRNA acceptor arm shuttling directly into the ATT active site without release\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. This channeling mode may help fungi produce Erg-Asp efficiently and independently from protein synthesis, but its molecular details remain unknown. Importantly, ErdS is phylogenetically related to bacterial aaPGSs\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e, but aminoacylates sterols instead of glycerolipids, implying that the \u003cem\u003ed\u003c/em\u003eGNAT module of ErdS contains an undescribed sterol-recognition fold.\u003c/p\u003e\u003cp\u003eIn fungi, Erg plays a crucial role in maintaining the structure and function of cell membranes, thereby directly affecting fungal viability and pathogenicity\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Aspartylation of Erg by ErdS alters its physical and chemical properties, which might affect membranes and modify the responsiveness of cells to external stress, including to antimicrobials\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. However, due to its recent discovery, the functions of Erg-aa in fungi remain elusive. We recently determined that Erg-Asp influences the regulation of asexual sporulation (conidiation) in \u003cem\u003eAspergillus oryzae\u003c/em\u003e (\u003cem\u003eAor\u003c/em\u003e), whereas Erg-Gly seems to promote the production of aerial hyphae\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Both Erg-aa slightly modify the resistance of \u003cem\u003eAor\u003c/em\u003e towards two antifungal drugs, suggesting a direct influence on membrane properties and/or indirect regulatory effects. ErdS is also present in a plethora of pathogenic fungi, such as \u003cem\u003eAfm\u003c/em\u003e, which can cause aspergillosis in immuno-compromised individuals\u0026mdash;a life threatening condition associated with poor prognosis\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e\u0026mdash;and \u003cem\u003eMagnaporthe oryzae\u003c/em\u003e (\u003cem\u003eMor\u003c/em\u003e) (rice blast fungus), a cereal pathogen that threatens global agriculture\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e, but the influence of Erg-Asp on their physiology remains unexplored.\u003c/p\u003e\u003cp\u003eHere, we show that Erg-Asp participates in asexual sporulation in \u003cem\u003eAfm\u003c/em\u003e and \u003cem\u003eMor\u003c/em\u003e. In \u003cem\u003eAfm\u003c/em\u003e, the deletion of the \u003cem\u003eerdS\u003c/em\u003e gene alters the germination rate of conidia, whereas its deregulation or overexpression increases the production of aerial hyphae and delays conidiation. ErdS and the Erg-Asp hydrolase, ErdH, seem to be expressed at all stages of the \u003cem\u003eAfm\u003c/em\u003e life cycle and dynamically localized over the course of fungal development. The deletion of \u003cem\u003eerdS\u003c/em\u003e also affects the resistance to high salt stress and the cell wall-targeting compound Congo Red, suggesting that Erg-Asp might participate in a vast array of processes. In this context, and because ErdS presents unique structural features, deciphering the molecular underpinnings of these tRNA-mediated modifications would attract both therapeutic and agricultural interest\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e,\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. Using cryo-electron microscopy (cryo-EM), we determined the structures of the \u003cem\u003eAfm\u003c/em\u003e ErdS apoenzyme and its complex with tRNA. The structures reveal a novel sterol binding pocket and uncover a unique mechanism in which the AspRS long N-terminal α-helix sequesters tRNA\u003csup\u003eAsp\u003c/sup\u003e, thereby facilitating Asp channeling between the AspRS and ATT active sites, with the RNA moiety acting in a manner mimicking swinging-arm prosthetic cofactors. This mode of tRNA capture might facilitate Erg-Asp synthesis at appropriate locations in a context where ErdS is dynamically localized during fungal development\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eErdS and ErdH are involved in asexual sporulation\u003c/h2\u003e\u003cp\u003eTo obtain insights into the physiological influence of Erg-Asp, we employed two fungal models: the human opportunistic pathogen \u003cem\u003eAfm\u003c/em\u003e and the plant pathogen \u003cem\u003eMor\u003c/em\u003e. We used the previously described \u003cem\u003e∆erdS\u003c/em\u003e mutant\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e of \u003cem\u003eAfm\u003c/em\u003e, which does not produce Erg-Asp. Despite our best efforts, we could not obtain a Δ\u003cem\u003eerdH\u003c/em\u003e mutant of \u003cem\u003eAfm\u003c/em\u003e that accumulated Erg-Asp. We circumvented this issue by reintroducing the \u003cem\u003eerdS\u003c/em\u003e gene at its locus under the control of the xylose promoter (P\u003csub\u003e\u003cem\u003eXyl\u003c/em\u003e\u003c/sub\u003e) in the \u003cem\u003e∆erdS\u003c/em\u003e strain (\u003cem\u003e∆erdS\u003c/em\u003e::P\u003csub\u003e\u003cem\u003eXyl\u003c/em\u003e\u003c/sub\u003e\u003cem\u003e-erdS\u003c/em\u003e strain). We also constructed a WT strain in which the P\u003csub\u003e\u003cem\u003eXyl\u003c/em\u003e\u003c/sub\u003e\u003cem\u003e-erdS\u003c/em\u003e construct directly replaced the original \u003cem\u003eerdS\u003c/em\u003e copy (WT::P\u003csub\u003e\u003cem\u003eXyl\u003c/em\u003e\u003c/sub\u003e\u003cem\u003e-erdS\u003c/em\u003e strain). Erg-Asp strongly accumulates in those strains when P\u003csub\u003e\u003cem\u003eXyl\u003c/em\u003e\u003c/sub\u003e\u003cem\u003e-erdS\u003c/em\u003e is induced with xylose (Xyl) and is barely synthesized under repressive conditions in the presence of glucose (Glc) (\u003cb\u003eFig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA\u003c/b\u003e), confirming the deregulation of ErdS expression.\u003c/p\u003e\u003cp\u003eWhen \u003cem\u003eAfm\u003c/em\u003e conidia (asexual spores) are point-inoculated on plates, they form round-shaped filamentous colonies with green conidia in the oldest areas, and the maturing and actively growing mycelia form a thin white circular margin (\u003cb\u003eFig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eB and S1C\u003c/b\u003e). On rich medium (malt agar), colonies of WT and Δ\u003cem\u003eerdS\u003c/em\u003e strains showed no obvious morphological differences, whereas both P\u003csub\u003e\u003cem\u003eXyl\u003c/em\u003e\u003c/sub\u003e\u003cem\u003e-erdS\u003c/em\u003e strains (\u003cem\u003e∆erdS\u003c/em\u003e::P\u003csub\u003e\u003cem\u003eXyl\u003c/em\u003e\u003c/sub\u003e\u003cem\u003e-erdS\u003c/em\u003e and WT::P\u003csub\u003e\u003cem\u003eXyl\u003c/em\u003e\u003c/sub\u003e\u003cem\u003e-erdS\u003c/em\u003e) presented a strong \u0026ldquo;fluffy\u0026rdquo; phenotype\u0026mdash;dense aerial hyphae\u0026mdash; under either repressing or inducing conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB-\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD), with much larger white margins and smaller conidiation areas. Colonies of all strains presented comparable diameters (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB), suggesting that the larger margins reflected a delay in conidiation. Since rich media can interfere with repression or induction of the P\u003csub\u003e\u003cem\u003eXyl\u003c/em\u003e\u003c/sub\u003e promoter, we point-inoculated conidia on minimal media (MM) supplemented with or without Glc or Xyl (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC, \u003cb\u003eFig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eD\u003c/b\u003e). Both P\u003csub\u003e\u003cem\u003eXyl\u003c/em\u003e\u003c/sub\u003e\u003cem\u003e-erdS\u003c/em\u003e strains again presented a strong fluffy phenotype under repressing (Glc) conditions, which was reduced\u0026mdash;although still visible\u0026mdash;under inducing conditions (Xyl), when ErdS was over-expressed. The P\u003csub\u003e\u003cem\u003eXyl\u003c/em\u003e\u003c/sub\u003e\u003cem\u003e-erdS\u003c/em\u003e strains also presented far fewer conidiophores than WT or Δ\u003cem\u003eerdS\u003c/em\u003e on the colony edges under repressing conditions, as observed by magnification (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, \u003cb\u003e3\u003c/b\u003erd row, \u003cb\u003eFig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eD\u003c/b\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eWe next spread conidia of the four strains on MM (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD) and observed the resulting mycelia lawns. The P\u003csub\u003e\u003cem\u003eXyl\u003c/em\u003e\u003c/sub\u003e\u003cem\u003e-erdS\u003c/em\u003e strains presented the fluffy phenotype at 20 h under either inducing or repressing conditions, and in contrast to the WT and Δ\u003cem\u003eerdS\u003c/em\u003e strains, no obvious conidiation (greenish-colored mycelium) was visible with the naked eye. Magnification confirmed that the WT and Δ\u003cem\u003eerdS\u003c/em\u003e strains produced a high density of conidiophores, while the P\u003csub\u003e\u003cem\u003eXyl\u003c/em\u003e\u003c/sub\u003e\u003cem\u003e-erdS\u003c/em\u003e strains formed very few (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD, rows 1 and 2). As in rich media, the induction of \u003cem\u003eerdS\u003c/em\u003e expression (Xyl) did not seem to reverse these phenotypes, likely because the density of mycelia from the convergence of thousands of micro-colonies in lawns exacerbates the remaining fluffy morphology. After 48 h (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD, \u003cb\u003e3\u003c/b\u003erd and 4th rows), conidiation eventually occurred in the P\u003csub\u003e\u003cem\u003eXyl\u003c/em\u003e\u003c/sub\u003e\u003cem\u003e-erdS\u003c/em\u003e strains, but the conidiophores were embedded in fluffy aerial hyphae (\u003cb\u003eFig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eE\u003c/b\u003e). This confirmed that changing the regulation of \u003cem\u003eerdS\u003c/em\u003e by placing it under the control of another promoter leads to delayed conidiation.\u003c/p\u003e\u003cp\u003eIn \u003cem\u003eMor\u003c/em\u003e, we could delete \u003cem\u003eerdS\u003c/em\u003e (\u003cem\u003e∆erdS\u003c/em\u003e, no Erg-Asp synthesis) and \u003cem\u003eerdH\u003c/em\u003e (\u003cem\u003e∆erdH\u003c/em\u003e, no Erg-Asp hydrolase activity) (\u003cb\u003eFig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eF\u003c/b\u003e). We noted that the WT strain produced undetectable levels of Erg-Asp (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE, \u003cem\u003eleft panel\u003c/em\u003e), as we had also observed in \u003cem\u003eNeurospora crassa\u003c/em\u003e (\u003cem\u003eNcr\u003c/em\u003e)\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE, \u003cem\u003eright panel\u003c/em\u003e), another Sordariomycete. As in \u003cem\u003eNcr\u003c/em\u003e, the deletion of \u003cem\u003eerdH\u003c/em\u003e resulted in Erg-Asp accumulation, although at low levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). Interestingly, in contrast to \u003cem\u003eAfm\u003c/em\u003e, the deletion of \u003cem\u003eerdS\u003c/em\u003e triggered a one order of magnitude decrease in conidiation (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF), which parallels the phenotype observed in \u003cem\u003eAor\u003c/em\u003e for the same mutant\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Colonies of both \u003cem\u003eMor\u003c/em\u003e mutants presented a larger white mycelium margin, although it was less prominent than those observed in \u003cem\u003eAfm\u003c/em\u003e P\u003csub\u003e\u003cem\u003eXyl\u003c/em\u003e\u003c/sub\u003e\u003cem\u003e-erdS\u003c/em\u003e strains, also reflecting delayed conidiation throughout colony development. Surprisingly, the \u003cem\u003eerdH\u003c/em\u003e deletion fully phenocopied that of \u003cem\u003eerdS\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF), suggesting that appropriately regulated levels of Erg-Asp are not only required for proper conidiation timing and/or levels in \u003cem\u003eMor\u003c/em\u003e as well as in \u003cem\u003eAfm\u003c/em\u003e, but also for normal hyphal growth in the latter.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eErg-Asp influences germination rate\u003c/h3\u003e\n\u003cp\u003eBeyond hyphal growth and conidiation, another important aspect of \u003cem\u003eAfm\u003c/em\u003e physiology is the germination of conidia. We measured the germination kinetics of WT, Δ\u003cem\u003eerdS\u003c/em\u003e, and \u003cem\u003e∆erdS\u003c/em\u003e::P\u003csub\u003e\u003cem\u003eXyl\u003c/em\u003e\u003c/sub\u003e\u003cem\u003e-erdS\u003c/em\u003e conidia (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA) and observed that the initial isotropic swelling proceeded normally in all cases, with normal proportions of swollen spores in the three strains. However, the emergence of germ tubes was slower with the \u003cem\u003e∆erdS\u003c/em\u003e swollen conidia (mean rate 26.0% h\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) compared to WT conidia (49.5% h\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e). As a result, at 9 h, most WT conidia had advanced, as expected, to hyphal extension, while\u0026thinsp;~\u0026thinsp;25% of Δ\u003cem\u003eerdS\u003c/em\u003e conidia remained non-germinated. For \u003cem\u003e∆erdS\u003c/em\u003e::P\u003csub\u003e\u003cem\u003eXyl\u003c/em\u003e\u003c/sub\u003e\u003cem\u003e-erdS\u003c/em\u003e conidia, germination started only after 6 hours (3\u0026ndash;4 h for WT conidia) post-inoculation, and the germination rate could not be evaluated under these conditions. This nevertheless suggested that the lack of Erg-Asp synthesis either delays the germination of \u003cem\u003eAfm\u003c/em\u003e conidia or alters synchronous germination\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e,\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eOther types of sterol conjugations are known to impact the immune recognition of fungal spores\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. We thus decided to monitor the phagocytosis of FITC-stained dormant or swollen conidia by IC-21 murine peritoneal macrophages (IC-21 MPM), using fluorescence microscopy (\u003cb\u003eFig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eA\u003c/b\u003e). We did not observe significant differences in adhesion to or internalization by macrophages between \u003cem\u003eAfm\u003c/em\u003e WT, ∆\u003cem\u003eerdS\u003c/em\u003e or \u003cem\u003e∆erdS\u003c/em\u003e::P\u003csub\u003e\u003cem\u003eXyl\u003c/em\u003e\u003c/sub\u003e\u003cem\u003e-erdS\u003c/em\u003e conidia, suggesting that Erg-Asp has no major role in the recognition by these immune cells. The same conclusion was drawn from Nanolive microscopy (\u003cb\u003eFig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eB\u003c/b\u003e, \u003cb\u003eExtended movies 1 and 2\u003c/b\u003e). However, the Kaplan-Meier curves representing the proportion of non-germinated conidia (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB) confirmed that the proportion of non-germinated conidia dropped sharply before 6 h with the WT strain (Multiplicities of infection [MOIs] of 5 and 10 showed no significant difference, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.32, see \u003cb\u003eFig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eB\u003c/b\u003e for statistics), with \u0026gt;\u0026thinsp;80% of germinated spores at 7\u0026ndash;8 h post-inoculation (6\u0026ndash;7 h after recording started). However, the Δ\u003cem\u003eerdS\u003c/em\u003e conidia exhibited slower germination rates at an MOI of 5, and especially at an MOI of 10 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). In addition, the final proportion of germinated conidia reached\u0026thinsp;~\u0026thinsp;60% (MOI of 5) or only\u0026thinsp;~\u0026thinsp;30% (MOI of 10) for \u003cem\u003e∆erdS\u003c/em\u003e conidia. This indicated that either the germination success was lower for the mutant, and/or the MOI had an influence on germination. Higher conidial densities are known to decrease germination rates\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e, but here the Δ\u003cem\u003eerdS\u003c/em\u003e mutation or Erg-Asp synthesis deregulation (\u003cem\u003e∆erdS\u003c/em\u003e::P\u003csub\u003e\u003cem\u003eXyl\u003c/em\u003e\u003c/sub\u003e\u003cem\u003e-erdS\u003c/em\u003e) might amplify this phenomenon, or perturb synchronous germination within the population.\u003c/p\u003e\u003cp\u003e\u003cb\u003eDynamic localizations of ErdS and ErdH in\u003c/b\u003e \u003cb\u003eA. fumigatus\u003c/b\u003e\u003c/p\u003e\u003cp\u003eGiven that ErdS and ErdH\u0026mdash;and thus Erg-Asp levels\u0026mdash;seem to impact conidiation, germination, and hyphal growth, we analyzed their subcellular localizations throughout the fungal life cycle (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). We thus replaced the \u003cem\u003eerdH\u0026mdash;erdS\u003c/em\u003e bigenic cluster of \u003cem\u003eAfm\u003c/em\u003e with a version enabling the expression of ErdS and ErdH C-terminally fused to eGFP and mCherry, respectively. Fluorescence microscopy showed that that \u003cem\u003eAfm\u003c/em\u003e ErdS and ErdH are both expressed at all stages of the fungal life cycle and already present in resting spores\u0026mdash;consistent with the observation of Erg-Asp in total lipids extracted from dormant conidia (\u003cb\u003eFig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eC\u003c/b\u003e).\u003c/p\u003e\u003cp\u003eIn resting conidia, ErdS-eGFP is diffusely localized in the cytosol, while ErdH-mCherry is concentrated in cytosolic round-shaped structures that only partially colocalize with ErdS-eGFP (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, t\u0026thinsp;=\u0026thinsp;0). In swollen conidia (\u003cem\u003et\u003c/em\u003e\u0026thinsp;=\u0026thinsp;3 h), a portion of the ErdS-eGFP starts to concentrate in puncta-like patches that colocalize with ErdH-mCherry puncta. In germinating conidia (\u003cem\u003et\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6 h) most of the ErdS-eGFP and ErdH-mCherry signals display granular distribution patterns that, on average, do not seem to colocalize. ErdS-eGFP seems evenly distributed and diffuse in the cytosol of growing mycelia, while ErdH is enriched on or within structures that resemble vacuoles (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, t\u0026thinsp;=\u0026thinsp;16 h); however, fractions of both enzymes also appear to colocalize at the plasma membrane, where most of the cellular aspartylable Erg resides. This localization is consistent with previous subcellular fractionation experiments on mycelia\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Finally, ErdS-eGFP and ErdH-mCherry are highly enriched at the plasma membranes of remnant germinating structures (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, t\u0026thinsp;=\u0026thinsp;16 h).\u003c/p\u003e\u003cp\u003eThese observations indicate that the localizations of ErdS and ErdH are highly dynamic throughout the fungal life cycle. It is therefore probable that Erg-Asp production temporally and spatially changes throughout the fungal lifecycle, and thus most likely regionally within colonies.\u003c/p\u003e\n\u003ch3\u003eErg-Asp might contribute to stress tolerance and cell wall integrity\u003c/h3\u003e\n\u003cp\u003eSince both ErdS and ErdH colocalize at some point in fungal development to the plasma membrane, this prompted us to test whether Erg-Asp could influence the sensitivity of \u003cem\u003eAfm\u003c/em\u003e to the cell wall disrupting agent Congo Red (CR). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD, the Δ\u003cem\u003eerdS\u003c/em\u003e strain was more sensitive to CR than the WT. This could indicate that the permeability of either the cell wall or plasma membrane\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e is affected in this mutant and that Erg-Asp might contribute to cell wall and/or plasma membrane integrity, at least under stressful conditions. Interestingly, the \u003cem\u003e∆erdS\u003c/em\u003e::P\u003csub\u003e\u003cem\u003eXyl\u003c/em\u003e\u003c/sub\u003e\u003cem\u003e-erdS\u003c/em\u003e strain partially recovered CR resistance on MM medium with glucose. In the absence of P\u003csub\u003e\u003cem\u003eXyl\u003c/em\u003e\u003c/sub\u003e\u003cem\u003e-erdS\u003c/em\u003e induction, the variability observed in complementation across replicates likely reflects the leakage due to the presence of the natural P\u003csub\u003e\u003cem\u003eErd\u003c/em\u003e\u003c/sub\u003e promoter upstream of P\u003csub\u003e\u003cem\u003eXyl\u003c/em\u003e\u003c/sub\u003e in our construct. However, this will require a more in-depth characterization that goes beyond the scope of this study. In contrast, osmotic stress did not produce significant growth differences (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE), but under high salt stress (NaCl and KCl), the WT strain presented conidiation defects (\u003cb\u003eFig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eD\u003c/b\u003e), whereas the Δ\u003cem\u003eerdS\u003c/em\u003e strain was less affected.\u003c/p\u003e\u003cp\u003eThe regulation of Erg-Asp synthesis and its levels under various conditions is currently unknown. The only condition under which we found a significant variation of Erg-Asp levels was when the WT \u003cem\u003eAfm\u003c/em\u003e strain, first grown in liquid MM containing a nitrogen (N) source (ammonium) was transferred for 5 h into nitrogen-free MM medium, which led to a\u0026thinsp;~\u0026thinsp;1.9-fold decrease in Erg-Asp within the total lipids, as judged by TLC (\u003cb\u003eFig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eE\u003c/b\u003e), suggesting that N availability might be a signal for Erg-Asp synthesis. However, we did not detect significant differences in growth rates between WT and Δ\u003cem\u003eerdS\u003c/em\u003e strains when grown in the presence of various N sources (\u003cb\u003eFig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eF\u003c/b\u003e) (ammonium, urea, bovine serum albumin, amino acids). When the N source was progressively reduced, however, the \u003cem\u003e∆erdS\u003c/em\u003e and \u003cem\u003e∆erdS\u003c/em\u003e::P\u003csub\u003e\u003cem\u003eXyl\u003c/em\u003e\u003c/sub\u003e\u003cem\u003e-erdS\u003c/em\u003e strains showed\u0026thinsp;~\u0026thinsp;1.70-fold increased growth rates under N deprivation (0 mM ammonium) or limitation (1 mM), compared to N replete (10 mM) conditions, that the WT strain did not exhibit. These differences tended to decrease under inducing conditions (MM\u0026thinsp;+\u0026thinsp;Xyl) in the \u003cem\u003e∆erdS\u003c/em\u003e::P\u003csub\u003e\u003cem\u003eXyl\u003c/em\u003e\u003c/sub\u003e\u003cem\u003e-erdS\u003c/em\u003e strain, suggesting the influence of Erg-Asp in this phenotype (\u003cb\u003eFig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eG\u003c/b\u003e).\u003c/p\u003e\u003cp\u003eGiven the diversity of effects induced by the absence and over-expression of ErdS in fungi, it seems that the temporally and spatially controlled synthesis of Erg-Asp is required for normal fungal development and propagation. This raised questions regarding how ErdS recognizes Erg and ensures proper Erg-Asp synthesis for normal germination, growth and conidiation, as well as when the fungus copes with stress. We thus performed a structural characterization of ErdS to better understand Erg-Asp synthesis in Dikarya.\u003c/p\u003e\u003cp\u003e\u003cb\u003eStructure and architecture of\u003c/b\u003e \u003cb\u003eAfm\u003c/b\u003e \u003cb\u003eErdS: the aa-tRNA binding site\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe closest homologs of ErdS in the \u003cem\u003ed\u003c/em\u003eGNATα\u003csup\u003e(+)\u003c/sup\u003e ATT family are the bacterial aaPGSs that aminoacylate glycerolipids, which suggests that their lipid-binding pockets\u0026mdash;which partly reside within the GNAT I subdomain\u0026mdash;should differ structurally. No known sterol-binding domain was detected in ErdS and, to our knowledge, no GNAT-related domain characterized thus-far has been reported to bind sterols\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. Similar to the other ATTs of the same structural fold, the GNAT II domain is expected to recognize the aa-tRNA moiety. Lastly, the mechanism by which the tRNA\u003csup\u003eAsp\u003c/sup\u003e acceptor stem travels between the AspRS and ATT active sites remains unknown. To identify the aa-tRNA and sterol binding pockets of ErdS, we expressed and purified an \u003cem\u003eAfm\u003c/em\u003e ErdS mutant lacking the N-terminal 86 residues (ErdSΔN1-86) for more efficient expression, mixed it with \u003cem\u003ein vitro\u003c/em\u003e-transcribed \u003cem\u003eAfm\u003c/em\u003e tRNA\u003csup\u003eAsp\u003c/sup\u003e and/or ergosterol, and performed single particle cryo-EM analyses.\u003c/p\u003e\u003cp\u003eThe first cryo-EM density map obtained showed that ErdSΔN1-86 bound neither tRNA\u003csup\u003eAsp\u003c/sup\u003e nor Erg, and that the apo-protein formed an homotetramer (\u003cb\u003eFig. \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e\u003c/b\u003e). This tetramer is composed of two ErdS dimers (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA) formed by the interactions between two AspRS domains, in a manner similar to that observed in the crystal structure of yeast AspRS\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Despite several attempts to obtain the structure of ErdS with bound Erg, we have not captured the sterol in the ATT domain, possibly due to its low solubility. No information on aa-tRNA or Erg binding could thus be obtained at this stage. Nevertheless, the structures of both AAT domains found in the ErdS dimer could be resolved (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Each has the \u003cem\u003ed\u003c/em\u003eGNATα\u003csup\u003e(+)\u003c/sup\u003e architecture consisting of two GNAT folds linked by a positively-charged α helix (noted α\u003csup\u003e(+)\u003c/sup\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, purple), similar to the architectures of the ATT domain of aaPGSs\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e and the bacterial Fem-ligases (FemX) (\u003cb\u003eFig. \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003eA\u003c/b\u003e) involved in the tRNA-dependent aminoacylation of the peptidoglycan precursor\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eIn the ErdS dimer, each ATT features a deep, large pocket in the GNAT II domain, next to the α\u003csup\u003e(+)\u003c/sup\u003e helix, with a surface predominantly covered in positively-charged or neutral residues (\u003cb\u003eFig. \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003eB\u003c/b\u003e), which likely corresponds to the Asp-tRNA-binding site. As expected, superimposition of the structure of the ErdS ATT domain onto that of FemX, complexed with a tRNA CCA-end analog\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e (\u003cb\u003eFig. \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003eC-4E\u003c/b\u003e), revealed that the CCA-end analog fit well at the entrance of this cavity (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC; \u003cb\u003eFig. \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003eA-4E\u003c/b\u003e). Moreover, in ErdS, W785 appears in the proximity of the A76 and C75 phosphate groups, with which it could interact \u003cem\u003evia\u003c/em\u003e anion-π interactions. R789 is positioned near the ribose of A76, and thus could interact \u003cem\u003evia\u003c/em\u003e hydrogen bond interactions (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo study the contributions of several residues of ErdS to the Erg-Asp synthesis activity, we constructed the corresponding mutants and expressed the ErdS variants in the yeast \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e (\u003cem\u003eSce\u003c/em\u003e)\u0026mdash;which possesses no ErxS homolog\u0026mdash;as previously reported\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. The Erg-Asp synthesis was then assessed by thin-layer chromatography (TLC) after lipid extraction. To facilitate comparisons, the Erg-Asp levels were normalized to those of phosphatidylethanolamine (Erg-Asp/PE ratio, noted \u003cem\u003eR\u003c/em\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). Of note, without Erg-Asp, phosphatidylglycerol becomes visible, which results in an \u003cem\u003eR\u003c/em\u003e ratio not equal to zero. First, we investigated the conserved GNAT II residues in the putative CCA pocket (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC) and the Lys/Arg residues located in the α\u003csup\u003e(+)\u003c/sup\u003e helix, which are expected to further bind the tRNA moiety. R749, K750, R752 and R753 were mutated to Ala (ErdSα\u003csup\u003e(0)\u003c/sup\u003e mutant) or Glu (ErdSα\u003csup\u003e(\u0026minus;)\u003c/sup\u003e) (see Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). As expected, ErdSα\u003csup\u003e(\u0026minus;)\u003c/sup\u003e and ErdSα\u003csup\u003e(0)\u003c/sup\u003e completely lost the Erg-Asp synthesis activity, with \u003cem\u003eR\u003c/em\u003e values (0.12\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05 and 0.17\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06, respectively) comparable to that measured for an ErdS ∆ATT mutant lacking the entire ATT domain (0.17\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06), and well below that of WT ErdS (\u003cem\u003eR\u003c/em\u003e\u0026thinsp;=\u0026thinsp;1.42\u0026thinsp;\u0026plusmn;\u0026thinsp;0.27) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). We previously observed identical effects when the α\u003csup\u003e(+)\u003c/sup\u003e helix of the ergosteryl-glycine synthase (ErgS)\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e was mutated. Single mutations in residues shaping the CCA pocket (W785H, W785A, R789A, K790E, G791A, Q793R, H795A, F913A and K916E) dramatically affected the ErdS activity, with all \u003cem\u003eR\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.5. These results are consistent with their predicted involvement in CCA and tRNA binding (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Interestingly, the K790E variant (\u003cem\u003eR\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.40\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08) is naturally present in \u003cem\u003eMor\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD), which might explain the low ErdS activity in this fungus when \u003cem\u003eerdH\u003c/em\u003e is deleted (no Erg-Asp hydrolase) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE).\u003c/p\u003e\n\u003ch3\u003eErdS GNAT I domain harbors a new sterol-binding fold\u003c/h3\u003e\n\u003cp\u003eWe next docked Erg within a putative sterol-binding cavity, which superimposes with the peptidoglycan-binding pockets of bacterial ATT homologs (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF). Erg fits well in this cavity, with a conformation consistent with favorable steric and electrostatic interactions to orient the 3β-OH group towards the terminal A76 of tRNA, to which Asp is esterified. A structural comparison with the PG-bound form of the homolog LysPGS\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e revealed, as expected, that the overall shape of the pocket differs substantially (\u003cb\u003eFig. \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003eF\u003c/b\u003e), indicating that this predicted sterol-binding pocket (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF) is indeed unique in the \u003cem\u003ed\u003c/em\u003eGNATα\u003csup\u003e(+)\u003c/sup\u003e ATT family and might constitute a new sterol-binding fold. Based on a multiple alignment of 98 ErdS orthologs, we selected 16 potentially critical residues within this cavity (Y637, T641, S642, T643, S644, W645, D647, R649, N677, E712, C728, E731, E798, W802, Q836 and K838) among which 13 are strictly or moderately conserved (\u003cb\u003eFig. S5\u003c/b\u003e). The Q836R mutation completely abolished the ErdS activity (\u003cem\u003eR\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.14\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05), while other mutations significantly reduced the Erg-Asp synthesis activity. The S644A mutation had the strongest effect (\u003cem\u003eR\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.5), while the W645A, D647R and K838E mutations had milder consequences (0.5\u0026thinsp;\u0026lt;\u0026thinsp;\u003cem\u003eR\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;1.0) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). Mutations of the remaining 11 residues had only slight or statistically non-significant effects. Since the ErdS variants were all expressed at comparable levels in \u003cem\u003eSce\u003c/em\u003e strains (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD, immunoblot), the decreased ErdS activities solely originate from the introduced mutations (with exception of F913A, which was expressed at low levels). We ruled out the possible long-range effects of these mutations on the AspRS activity\u0026mdash;that would also reduce Erg-Asp synthesis, even if Asp-tRNA can be scavenged from the cellular pool\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e\u0026mdash;since all of these ErdS variants could replace the endogenous \u003cem\u003eSce\u003c/em\u003e AspRS (Dps1) by complementation of a \u003cem\u003edps1∆\u003c/em\u003e strain\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e (\u003cb\u003eTable \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e\u003c/b\u003e). These results are in good agreement with our predicted location of the sterol-binding domain of ErdS.\u003c/p\u003e\n\u003ch3\u003eSterol aspartylation by ErdS is highly promiscuous\u003c/h3\u003e\n\u003cp\u003eWe previously observed that \u003cem\u003eAfm\u003c/em\u003e ErdS aspartylates cholesterol (Cho) in addition to Erg, despite the structural differences between the two sterols\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Consequently, Cho fits well within ErdS\u0026rsquo;s sterol pocket in docking models (\u003cb\u003eFig. S6A\u003c/b\u003e). Diosgenin, which features an unusual cyclized alkylyl side-chain (\u003cb\u003eFig. S6A\u003c/b\u003e), also seems to bind productively, suggesting that ErdS might have broad specificity for sterols. To investigate both the ErdS promiscuity towards sterols and the structural determinants for sterol recognition, we performed an \u003cem\u003ein vitro\u003c/em\u003e sterol aminoacylation assay\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e with a panel of structurally different sterols (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eH, \u003cb\u003eFig. S6B and 6C\u003c/b\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003eAfm\u003c/em\u003e ErdS aspartylates sterols regardless of the alkylyl side-chain\u0026rsquo;s structure, since in addition to Erg and Cho (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG, \u003cem\u003eleft panel\u003c/em\u003e, lanes 1, 2 and 4), it aspartylated the human hormones pregnenolone and \u003cem\u003etrans\u003c/em\u003e-dehydroandrosterone (DHA), although they present much smaller polar side-chains (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG, \u003cem\u003eleft panel\u003c/em\u003e, lanes 6 and 7). The lower aspartylation levels suggest that the alkylyl moiety could enhance the proper anchoring within the sterol pocket (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG). Consequently, a variety of other sterols can be used as substrates, despite the fact that they differ in the numbers (0, 1 or 2) and positions of saturations in the B-ring or in the bulkiness, saturation, and methylation pattern of the alkylyl side chain (\u003cb\u003eFig. S6B and S6C\u003c/b\u003e, lathosterol, zymosterol, β-sistosterol, stigmasterol, coprostanol, fucosterol, 5α-cholestan-3β-ol). As predicted above (\u003cb\u003eFig. S6A\u003c/b\u003e), diosgenin is efficiently aspartylated by ErdS, indicating that a cyclized side chain does not prevent recognition and aminoacylation. Lanosterol, which features a 4-dimethylated position in the A-ring of the cyclopentanophenantrene (sterane) nucleus, however, is not aspartylated \u003cem\u003ein vitro\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG, left panel, lane 3, \u003cb\u003eFig. S6B\u003c/b\u003e, lane 3), indicating that such bulky modifications may act as antideterminants for sterol recognition. Similarly, β-estradiol, with an aromatic A-ring, is not a substrate (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG, left panel, lane 8), despite its otherwise common features with DHA, showing that a non-aromatic ring is required.\u003c/p\u003e\u003cp\u003eInterestingly, coprostanol and epicoprostanol are identical, except that the first possesses a 3-OH group in the β configuration, whereas the second is in the α configuration. These two steroids and 5α-cholestan-3β-ol have the same alkylyl side chain as Cho (but no unsaturation at position 5 in the steran nucleus). As expected, coprostanol and 5α-cholestan-3β-ol are aspartylated at similar levels as Cho, despite the absence of the Δ5 unsaturation and their different absolute configurations at position C5 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG, \u003cem\u003eright panel\u003c/em\u003e). In contrast, epicoprostanol is not aminoacylated, most likely because the 3α-OH group is oriented away from the catalytic residues (see below) and the aminoacylated A76 residue of tRNA\u003csup\u003eAsp\u003c/sup\u003e. Consequently, cholic acid, which has a 3α-OH group, is also not a substrate (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG, \u003cem\u003eleft panel\u003c/em\u003e, lane 5).\u003c/p\u003e\u003cp\u003eThis promiscuous activity also exists \u003cem\u003ein vivo\u003c/em\u003e, as demonstrated by the heterologous expression of \u003cem\u003eAfm\u003c/em\u003e ErdS in \u003cem\u003eSce\u003c/em\u003e Erg biosynthesis pathway mutants\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e (\u003cem\u003eerg3\u003c/em\u003e∆, \u003cem\u003eerg4\u003c/em\u003e∆, \u003cem\u003eerg5\u003c/em\u003e∆ and \u003cem\u003eerg6\u003c/em\u003e∆), which accumulate various Erg intermediates (\u003cb\u003eFig. S6D and S6E\u003c/b\u003e). TLC analyses of total lipids from these strains revealed that ErdS aspartylates Erg intermediates that mostly differ in the saturation of the B-ring and the alkylyl side-chain, as expected from our \u003cem\u003ein vitro\u003c/em\u003e results; notably, the \u003cem\u003eerg6\u003c/em\u003eΔ strain accumulates zymosterol, which was also aminoacylated by ErdS \u003cem\u003ein vitro\u003c/em\u003e (\u003cb\u003eFig. S6B\u003c/b\u003e, lane 5). Therefore, ErdS is highly promiscuous for sterols, with the minimal requirements that the A-ring is non-aromatic and free of methyl groups and the 3-OH is in the β configuration.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eOrganization of the active site and catalytic mechanism of transesterification\u003c/h2\u003e\u003cp\u003eSimultaneous docking of Erg and the CCA analog revealed that the 3-OH group of Erg, when in the β configuration, is oriented towards the 2\u0026acute;-OH of the ribose moiety of A76, in a geometry suitable for transesterification (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE \u003cb\u003eand Fig. S6F-S6G\u003c/b\u003e). The α configuration evidently misorients the 3-OH group of Erg (or any sterol) within the active site, preventing it from reacting with the Asp-tRNA\u003csup\u003eAsp\u003c/sup\u003e ester bond. In FemX complexed to the CCA analog and a peptidoglycan precursor, the nucleophile is provided by the ε-amino group of a Lys side chain from the peptidoglycan precursor before transpeptidation. By contrast, Erg must first be activated by ErdS. We propose (\u003cb\u003eFig. S6F and S6G\u003c/b\u003e) that H795 acts as a general base to deprotonate the 3-OH group of Erg, thereby generating a nucleophilic alkoxide. This activated 3-alkoxide is positioned to attack the carbonyl carbon of the aminoacyl ester in Asp-tRNA\u003csup\u003eAsp\u003c/sup\u003e. During this reaction, the negative charge developing on the carbonyl oxygen is likely stabilized by the positively-charged guanidinium group of R914, facilitating the transesterification (\u003cb\u003eFig. S6G\u003c/b\u003e). The K305 residue of FemX has an equivalent function, and is located at the same position in superimposed models. This mechanism provides hints toward elucidating how the active site architecture of ErdS supports the selective transfer of the aspartyl group to only 3β-OH-containing sterol substrates. The presence of methyl groups at C4 of the sterol A-ring likely introduces steric hindrance, preventing proper accommodation within the binding pocket.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eThe N-terminal long α-helix of ErdS clamps the tRNA elbow to ensure enzymatic activity\u003c/h3\u003e\n\u003cp\u003eThe N-terminal extension in the Class IIb aaRSs of higher eukaryotes contains a conserved alpha-helix, which has been suggested to play a crucial role in enhancing their affinity for tRNA\u003csup\u003e\u003cspan additionalcitationids=\"CR38 CR39\" citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. Although the crystal structure of AspRS has been determined, this N-terminal region was deleted in the resolved structure, and thus requires further investigation to elucidate its role in tRNA binding\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. Notably, ErdS also possesses a similar N-terminal helix that includes the conserved xSKxxLKKxx motif (with the exception of \u003cem\u003eMor\u003c/em\u003e), which was previously proposed to be critical for tRNA binding\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e,\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e(Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). The recently reported cryo-EM structure of human LysRS\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e provides insights into the partial structure of this helix and its interactions with tRNA. However, the density corresponding to the anticodon-binding region remains unresolved, and its complete structural configuration awaits elucidation. To investigate the role of the N-terminal helix, we prepared full length ErdS (FL-ErdS), incubated it with \u003cem\u003ein vitro-\u003c/em\u003etranscribed \u003cem\u003eAfm\u003c/em\u003e tRNA\u003csup\u003eAsp\u003c/sup\u003e, and performed a cryo-EM analysis. The structure revealed the dimeric configuration of ErdS in complex with two tRNA\u003csup\u003eAsp\u003c/sup\u003e molecules (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB; \u003cb\u003eFigs. S8 and S9\u003c/b\u003e).\u003c/p\u003e\u003cp\u003eIn the complex, each protomer of the ErdS dimer binds one tRNA\u003csup\u003eAsp\u003c/sup\u003e molecule (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB-\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). The anticodon of tRNA\u003csup\u003eAsp\u003c/sup\u003e interacts with the anticodon-binding domain (ABD) of the AspRS domain, and the acceptor stem is positioned within the catalytic domain of AspRS, consistent with the previously reported crystal structure of the \u003cem\u003eSce\u003c/em\u003e AspRS/tRNA\u003csup\u003eAsp\u003c/sup\u003e complex\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. The ATT domain was disordered, suggesting that it moves flexibly relative to AspRS. Interestingly, residues 59\u0026ndash;102 of the NTD form an extended linear pair of α-helices separated by a connective loop, which spans from the anticodon to the elbow region of tRNA\u003csup\u003eAsp\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB-\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE). This N-terminal region is rich in polar amino acids, which form multiple interactions with the phosphate backbone, ribose moieties, and nucleotide bases of tRNA\u003csup\u003eAsp\u003c/sup\u003e. Residues R61 and N62 interact with the phosphate backbone of residues G50 to G52 in the T-stem. The highly conserved K65 interacts with the ribose of G57 in the T-loop. C20a of tRNA\u003csup\u003eAsp\u003c/sup\u003e is flipped outward, with its base forming a salt bridge with R69 of the NTD. Lastly, E70 and S77 interact with the phosphate backbone of tRNA\u003csup\u003eAsp\u003c/sup\u003e\u0026rsquo;s variable loop. Further stabilization is provided by E81, Q84, and D87 within the connective loop between the α-helices, which, along with H92, interact with either the ribose or the phosphate backbone of the anticodon stem.\u003c/p\u003e\u003cp\u003eWe previously reported that full-length \u003cem\u003eAfm\u003c/em\u003e ErdS can complement an \u003cem\u003eSce dps1\u003c/em\u003eΔ strain (AspRS gene deletion), although less efficiently than an \u003cem\u003eAfm\u003c/em\u003e ErdS ΔATT variant in which the ATT domain was removed, likely because \u003cem\u003eSce\u003c/em\u003e tRNA\u003csup\u003eAsp\u003c/sup\u003e was used primarily for Erg-Asp synthesis over protein synthesis in this context, which lacked extra AspRS\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Based on our cryo-EM structure, we hypothesized that the NTD could also contribute to tRNA\u003csup\u003eAsp\u003c/sup\u003e sequestering within ErdS, to ensure its dedicated participation in Erg-Asp synthesis. To ascertain the contribution of the observed N-terminal domain in tRNA\u003csup\u003eAsp\u003c/sup\u003e sequestering, we designed mutants with progressive NTD truncations (ErdSΔN30, ΔN60, and ΔN84, in which the first 30, 60 or 84 residues were removed, respectively) and tested their complementation efficiency in the \u003cem\u003edps1\u003c/em\u003eΔ strain by plasmid shuffling. First, we confirmed that the ErdS ΔATT mutant complemented the \u003cem\u003edps1\u003c/em\u003eΔ strain as efficiently as Dps1 (\u003cem\u003eSce\u003c/em\u003eAspRS). Strikingly, the growth progressively improved with the NTD truncations of \u003cem\u003eAfm\u003c/em\u003e ErdS, with the ΔN84 mutant behaving like the ErdS ΔATT mutant, as judged by the \u003cem\u003eSce\u003c/em\u003e colony sizes (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF) and by drop tests (\u003cb\u003eFig. S7\u003c/b\u003e). This indicates that the region between residues 60 and 84\u0026mdash;\u003cem\u003ei.e.\u003c/em\u003e, where the xSKxxLKKxx motif lies\u0026mdash;has the strongest contribution to growth reduction, and likely to tRNA\u003csup\u003eAsp\u003c/sup\u003e sequestering. The growth improvement was interpreted as the easier release of Asp-tRNA\u003csup\u003eAsp\u003c/sup\u003e from ErdS, in agreement with structural results.\u003c/p\u003e\u003cp\u003eIn parallel, we visualized Erg-Asp production in those strains and observed that Erg-Asp synthesis dropped by 17.8-, 15.0-, and 12.1-fold, respectively, for the ErdSΔN30, ΔN60, and ΔN84 mutants, indicating that the efficiency of Erg-Asp synthesis is affected by even the smallest truncation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF), and showing that the NTD is crucial for the full activity. In the case of \u003cem\u003eAor\u003c/em\u003e ErdS, the complementation of the \u003cem\u003edps1\u003c/em\u003eΔ strain was highly inefficient (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG), but the truncation of the NTD segment down to residue 90 (equivalent to Δ84N in \u003cem\u003eAfm\u003c/em\u003e ErdS) restored WT growth. Again, Erg-Asp synthesis was reduced (2.9-fold) in this mutant, compared to the full-length enzyme. These results suggest that, apart from the ATT domain, the NTD significantly contributes to tRNA\u003csup\u003eAsp\u003c/sup\u003e utilization in the \u003cem\u003ein vivo\u003c/em\u003e context, likely through sequestering it within ErdS, making it unavailable for protein synthesis and thereby reducing growth, while strongly increasing the efficiency of Erg-Asp synthesis. This explains why all ErdS-containing fungi have a second, separated AspRS\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e (ref PNAS and Ref JBC) that ensures a sufficient supply of Asp-tRNA\u003csup\u003eAsp\u003c/sup\u003e for protein synthesis.\u003c/p\u003e\n\u003ch3\u003etRNA behaves like a prosthetic swinging arm during the multi-enzymatic process of ErdS\u003c/h3\u003e\n\u003cp\u003eThe cryo-EM structural analysis of the ErdS/tRNA\u003csup\u003eAsp\u003c/sup\u003e complex revealed 3D classes with density suggestive of the ATT domain. Further analysis using a mask covering these weak densities improved the map, confirming that they extended from the C-termini of both AspRS domains, which validated them as the ATT domain (Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eB and S8C). In one ErdS protomer (ErdS1), the ATT1 module is positioned adjacent to the elbow of the tRNA\u003csub\u003e1\u003c/sub\u003e\u003csup\u003eAsp\u003c/sup\u003e bound to the corresponding AspRS1 domain, on the same side where the NTD1 also interacts along the anticodon stem and the elbow region. Interestingly, tRNA\u003csub\u003e1\u003c/sub\u003e\u003csup\u003eAsp\u003c/sup\u003e is not only supported at the elbow region by the ATT1 domain and the NTD1, but its CCA end is located near the ATT2 module from the other protomer (ErdS2), effectively sandwiching tRNA\u003csub\u003e1\u003c/sub\u003e\u003csup\u003eAsp\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). This explains why the \u003cem\u003edps1\u003c/em\u003eΔ strain expressing WT \u003cem\u003eAfm\u003c/em\u003e ErdS grew more slowly than the strains expressing Dps1 (\u003cem\u003eSce\u003c/em\u003e AspRS) or the \u003cem\u003eAfm\u003c/em\u003e ErdS ΔATT mutant (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF). ErdS predominantly directs Asp-tRNA\u003csup\u003eAsp\u003c/sup\u003e towards Erg-Asp synthesis\u0026mdash;resulting in slower growth in the absence of an independent AspRS\u0026mdash;because both the NTD and ATTs prevent its aminoacylated tRNA\u003csup\u003eAsp\u003c/sup\u003e from being released. In light of this structural information, we propose that the ATT domains of ErdS dimers cooperate not only in Erg-Asp synthesis but also, together with the NTD, in sequestering tRNA\u003csup\u003eAsp\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo further investigate the interaction between the ATT domain and the CCA end of tRNA\u003csup\u003eAsp\u003c/sup\u003e, we attempted the cryo-EM structural analysis of ErdS bound to Asp-tRNA. We reasoned that the ATT domain requires the Asp moiety to bind tRNA\u003csup\u003eAsp\u003c/sup\u003e over the AspRS domain, which in contrast recognizes uncharged tRNA\u003csup\u003eAsp\u003c/sup\u003e. Since aa-tRNAs are unstable, we prepared a non-hydrolyzable Asp-tRNA\u003csup\u003eAsp\u003c/sup\u003e analog (Asp-\u003cem\u003eN\u003c/em\u003e-tRNA\u003csup\u003eAsp\u003c/sup\u003e), by using a modified tRNA\u003csup\u003eAsp\u003c/sup\u003e transcript in which the 3\u0026acute;-hydroxy group of the terminal ribose is replaced by a 3\u0026acute;-amino group, so that the ester bond between Asp and tRNA\u003csup\u003eAsp\u003c/sup\u003e is replaced by an amido bond upon Flexizyme-mediated \u003cem\u003ein vitro\u003c/em\u003e aspartylation\u003csup\u003e\u003cspan additionalcitationids=\"CR44\" citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. We also prepared an ErdS variant with nine mutations in the AspRS domain (Q334E, S335A, N362P, S363A, N364P, H366A, R367A, H368A, and Y494F) to reduce its tRNA binding affinity and allow the Asp-CCA end to enter the ATT catalytic site. We then incubated Asp-\u003cem\u003eN\u003c/em\u003e-tRNA\u003csup\u003eAsp\u003c/sup\u003e with this purified ErdS variant and performed a cryo-EM structural analysis. This revealed the structure of an ErdS dimer bound with only one Asp-\u003cem\u003eN\u003c/em\u003e-tRNA\u003csup\u003eAsp\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eB, \u003cb\u003eFig. S10\u003c/b\u003e). This Asp-\u003cem\u003eN\u003c/em\u003e-tRNA\u003csub\u003e1\u003c/sub\u003e\u003csup\u003eAsp\u003c/sup\u003e was bound to the ABD of AspRS1 in a manner identical to that in the WT ErdS/tRNA\u003csup\u003eAsp\u003c/sup\u003e complex (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB and Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Surprisingly, in this case the CCA end protruded away from the AspRS1 active site (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eB to \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eE, \u003cb\u003eFig. S11\u003c/b\u003e). This structural rearrangement is accompanied by a 23 \u0026Aring; movement of the tRNA\u003csup\u003eAsp\u003c/sup\u003e CCA end and an 8 \u0026Aring; shift of the tRNA elbow (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). The ATT1 domain-binding Asp-\u003cem\u003eN\u003c/em\u003e-tRNA\u003csup\u003eAsp\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eB) was positioned similarly to that observed in the previous ErdS/tRNA\u003csup\u003eAsp\u003c/sup\u003e complex (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). It also interacted with the elbow of tRNA\u003csup\u003eAsp\u003c/sup\u003e, whereas the ATT2 domain from the tRNA\u003csup\u003eAsp\u003c/sup\u003e-free protomer (ATT2) showed no detectable density.\u003c/p\u003e\u003cp\u003eSurprisingly, these orientations of the acceptor stem and the CCA end of Asp-\u003cem\u003eN\u003c/em\u003e-tRNA\u003csub\u003e1\u003c/sub\u003e\u003csup\u003eAsp\u003c/sup\u003e did not direct them towards the active site (CCA pocket) of ATT1, raising questions on the mechanism by which the Asp moiety could be used to modify Erg. We aligned the tRNA\u003csup\u003eAsp\u003c/sup\u003e-free AspRS1 domain with the AspRS domain of the ErdS/tRNA\u003csup\u003eAsp\u003c/sup\u003e complex characterized previously, in order to determine the positions of the missing tRNA\u003csub\u003e2\u003c/sub\u003e\u003csup\u003eAsp\u003c/sup\u003e and the ATT\u003csub\u003e2\u003c/sub\u003e module. These models revealed that the CCA end of Asp-\u003cem\u003eN\u003c/em\u003e-tRNA\u003csub\u003e1\u003c/sub\u003e\u003csup\u003eAsp\u003c/sup\u003e was even closer to the ATT2 domain (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). Notably, the α\u003csup\u003e(+)\u003c/sup\u003e helix of ATT2, which was predicted to interact with the tRNA\u003csup\u003eAsp\u003c/sup\u003e acceptor arm, was the closest to the CCA end (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eE). The W785 and K916 residues, which are crucial for the CCA interaction and the activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA to \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD), were also close to the CCA end. These results suggest that, in the ErdS dimer, after aspartylation by the AspRS1 domain, the CCA end of tRNA\u003csub\u003e1\u003c/sub\u003e\u003csup\u003eAsp\u003c/sup\u003e swings to the ATT2 domain of the other protomer, allowing the efficient aminoacylation of Erg (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Since tRNA\u003csup\u003eAsp\u003c/sup\u003e seems to be trapped by its interactions with the NTD and both ATT domains, it is likely that it does not dissociate from ErdS, and that the acceptor arm shuttles back and forth from one active site (AspRS1) to the next (ATT2), serving as a prosthetic swinging arm that shuttles an activated Asp moiety for Erg-Asp synthesis. This fully explains the low capacity of ErdS to replace the endogenous AspRS in \u003cem\u003eSce\u003c/em\u003e, unless either the ATT or NTD domain is removed to facilitate Asp-tRNA\u003csup\u003eAsp\u003c/sup\u003e release from ErdS to promote protein synthesis.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eOur study reveals a distinct mechanism by which an aa-tRNA is employed for small-molecule aminoacylation in eukaryotes. Through structural and biochemical analyses, we show how ErdS catalyzes the transfer of aspartate from Asp-tRNA\u003csup\u003eAsp\u003c/sup\u003e to the 3β-OH group of Erg\u0026mdash;a lipid central to fungal membrane physiology. This reaction is enabled by a previously uncharacterized sterol-binding pocket within the ATT domain and a tRNA-mediated handover mechanism that coordinates two spatially separate active sites. These findings provide new insights into how enzymes can co-opt aa-tRNAs to expand their catalytic repertoire beyond and independently from translation.\u003c/p\u003e\u003cp\u003eErdS does not rely on the diffusion of intermediates. Instead, it uses the tRNA as a dynamic tether to direct the flow of the aminoacyl (aspartyl) group. First, the RNA moiety of the ErdS/tRNA\u003csup\u003eAsp\u003c/sup\u003e complex is sequestered within the ribonucleic particle, not only through interactions with the two ATT domains from the two ErdS protomers, but also by a long NTD that further interacts with the anticodon and elbow regions. This reinforces the interaction of the molecule with the AspRS part, thus stabilizing the tRNA through extensive interactions and guiding its movement across the enzyme, which likely prevents its release upon aspartylation, as suggested by complementation experiments in \u003cem\u003eSce\u003c/em\u003e. The acceptor arm and CCA end of tRNA\u003csup\u003eAsp\u003c/sup\u003e can thus undergo a large-scale repositioning (~\u0026thinsp;23 \u0026Aring;) to reach the ATT domain, while the domain itself is flexibly rearranged to facilitate this interaction. Unexpectedly, in the ErdS protomer, the tRNA acceptor arm does not simply shuttle from the AspRS active site to its appended (\u003cem\u003ecis\u003c/em\u003e) ATT domain, as we initially thought (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Instead, the \u003cem\u003ecis\u003c/em\u003e ATT functions as a stabilizer of the ErdS/tRNA\u003csup\u003eAsp\u003c/sup\u003e interaction, whereas the second ATT domain, situated in the second ErdS protomer, captures the CCA-end of tRNA\u003csup\u003eAsp\u003c/sup\u003e through interactions with the α\u003csup\u003e(+)\u003c/sup\u003e helix, and functions as a \u003cem\u003etrans\u003c/em\u003e-acting enzyme, using the aspartyl moiety to transfer it onto Erg. In this context, the tRNA functions as an architectural bridge\u0026mdash;dynamically connecting the AspRS and ATT domains\u0026mdash;in a manner reminiscent of the swinging arms seen in multienzyme complexes\u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e and of the shuttles between aminoacylation and amino acid editing functions observed in aminoacyl-tRNA synthetases\u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e,\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e\u003cp\u003eSchematics of this molecular choreography are depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e6\u003c/span\u003e and \u003cb\u003eExtended Data Movie 3\u003c/b\u003e.\u003c/p\u003e\u003cp\u003eThe domain architecture of ErdS, which fuses AspRS with a \u003cem\u003ed\u003c/em\u003eGNAT-type ATT module, is essential for this coordinated reaction. Our structural and mutational analyses suggest that the extended NTD, which differs from those found in canonical cytoplasmic AspRSs, plays a critical role in clamping the tRNA to stably position it for productive handover. Shortening of the NTD reduces the Erg-Asp synthesis efficiency \u003cem\u003ein vivo\u003c/em\u003e in the yeast heterologous model, and we previously observed that separating the AspRS and ATT domains also reduces the activity, including when the ATT domain is expressed alone in \u003cem\u003eAor\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e, illustrating the catalytic advantages of a single bifunctional enzyme. This integration of synthetase and transferase activities into a single polypeptide represents a functional innovation, enabling direct control over substrate transfer and reactivity. Our complementation experiments in \u003cem\u003eSce\u003c/em\u003e illustrated that ErdS poorly releases Asp-tRNA\u003csup\u003eAsp\u003c/sup\u003e, which is thus efficiently trapped throughout the Erg-Asp synthesis cycle, leading to decreased protein synthesis. Hence, in nature, Dikarya harboring ErdS always possess a second, canonical AspRS to ensure efficient protein synthesis\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Sequestering of tRNA\u003csup\u003eAsp\u003c/sup\u003e within ErdS also provides an advantage by diverting a subset of tRNA\u003csup\u003eAsp\u003c/sup\u003e from the cytosolic pool and dedicating it solely to Erg-Asp synthesis. This differs from free-standing ATTs, such as the Erg-Gly synthase also present in fungi, which rely on an independent aa-tRNA synthetase\u0026mdash;and therefore on protein synthesis\u0026mdash;to produce their aa-tRNA substrate in the context of competition with elongation factors fueling translating ribosomes with aa-tRNAs. The fusion between the AspRS and ATT domains in ErdS and the molecular mechanism described above may ensure proper Erg-Asp levels under challenging conditions and at dedicated subcellular localizations, to enable proper mycelium development, conidiation, and likely cell wall integrity, independently from protein synthesis.\u003c/p\u003e\u003cp\u003eThe deletion of \u003cem\u003eerdS\u003c/em\u003e in \u003cem\u003eAfm\u003c/em\u003e, \u003cem\u003eAor\u003c/em\u003e, or \u003cem\u003eMor\u003c/em\u003e leads to contrasting phenotypes. Coniditation is strongly reduced in Δ\u003cem\u003eerdS\u003c/em\u003e mutants of \u003cem\u003eAor\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e or \u003cem\u003eMor\u003c/em\u003e (this study), but not in \u003cem\u003eAfm\u003c/em\u003e. This may reflect differences in regulatory circuits between species or in the\u0026mdash;direct or indirect\u0026mdash;contribution of Erg-Asp to such processes. Of note, in the Δ\u003cem\u003eerdS\u003c/em\u003e strain of \u003cem\u003eAfm\u003c/em\u003e, the \u003cem\u003eergS\u003c/em\u003e gene that encodes the Erg-Gly synthase (ErgS) is still present, and we cannot exclude the possibility that Erg-Gly has overlapping functions that could attenuate phenotypes. Conversely, \u003cem\u003eMor\u003c/em\u003e lacks ErgS. Moreover, the deletion of the Erg-Asp hydrolase gene \u003cem\u003eerdH\u003c/em\u003e phenocopies the Δ\u003cem\u003eerdS\u003c/em\u003e mutation in \u003cem\u003eMor\u003c/em\u003e, suggesting that the deregulation of Erg-Asp levels, rather than the absence of ErgS, plays significant roles in phenotypes. Nevertheless, the deregulation or impaired synthesis of Erg-Asp always affects conidiation to various degrees\u0026mdash;from decreased conidia production to differences in conidiation timing. In \u003cem\u003eAfm\u003c/em\u003e it also affects vegetative growth (aerial hyphae production) and spore germination, and likely stress resistance as well. These effects are expected to differ greatly between ErdS/H-containing fungi, as illustrated by the differences in Erg-Asp levels during vegetative growth between \u003cem\u003eAfm\u003c/em\u003e and \u003cem\u003eAor\u003c/em\u003e on the one hand (Erg-Asp synthesis) and \u003cem\u003eNcr\u003c/em\u003e and \u003cem\u003eMor\u003c/em\u003e on the other hand (no detectable Erg-Asp). This may also be amplified by the presence or absence of ErgS, and thus, of Erg-Gly. In addition, the spatial and temporal regulations of Erg-Asp synthesis might also differ, not only between fungal species but also at the colony and cell levels within each species. Overall, Erg-Asp synthesis (ErdS) and regulation (ErdH) likely contribute to the fitness of fungi, as supported by their conservation across fungal clades in Dikarya. Further work is needed to understand the contributions of Erg-aa to the pathogenicity and stress resistance of these species, and to clarify their functions in reproduction and growth.\u003c/p\u003e\u003cp\u003eThe sterol-binding pocket discovered in the ATT domain adopts a geometry and electrostatic environment tailored to accommodate the rigid structure of Erg. The deep, hydrophobic cavity lined with basic residues provides both shape and charge complementarity to the ligand. This binding mode is structurally distinct from lipid-interacting enzymes such as LysPGS, and illustrates how the \u003cem\u003ed\u003c/em\u003eGNAT fold can be repurposed for the selective recognition of sterol substrates. Given the essential role of Erg in fungal membrane integrity and drug resistance, this well-defined pocket may serve as a promising scaffold for antifungal drug development. It appears highly promiscuous for sterols, but it is worth mentioning that while Erg is the main sterol of numerous fungi\u0026mdash;including \u003cem\u003eAfm\u003c/em\u003e, \u003cem\u003eAor\u003c/em\u003e, \u003cem\u003eMor\u003c/em\u003e or \u003cem\u003eNcr\u003c/em\u003e\u0026mdash;different types of sterols are used in other species\u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e that also possess ErdS, such as the Erg-related sterols in Agaricomycotina and the 24-ethylcholesterol-family molecules in Pucciniomycotina. This apparent promiscuity may reflect this molecular diversity, and could result from ErdS\u0026rsquo;s evolutionary history within Dikarya.\u003c/p\u003e\u003cp\u003eBy integrating amino acid activation, tRNA coordination, and sterol modification within a single, spatially organized framework, ErdS exemplifies a catalytic strategy built on architectural precision and dynamic domain interplay. Rather than simply combining functions, ErdS choreographs the positioning of its substrates through tRNA-guided movements and domain rearrangements. This mechanism not only reveals a unique solution to non-ribosomal aminoacyl transfer, but also offers a design principle for engineering synthetic enzymes with programmable spatial control. The structurally resolved sterol-binding pocket further provides a tractable and selective platform with potential relevance to antifungal drug development.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003ch2\u003eAuthor information\u003c/h2\u003e\u003cp\u003eH.M. performed structural analyses with assistance from M.N., Y.K. and Y.I.\u003c/p\u003e\u003cp\u003eN.Y., N.M. and S.Z. conducted fungal genetics, phenotypic characterization, and biochemical assays, with assistance from B.S., F.F. and H.D.B.\u003c/p\u003e\u003cp\u003eL.S. and F.M. performed the Nanolive imaging experiments.\u003c/p\u003e\u003cp\u003eH.B.G. and Y.M.H. prepared the Asp-tRNA\u003csup\u003eAsp\u003c/sup\u003e analog used for structural analysis.\u003c/p\u003e\u003cp\u003eH.M. and F.F. wrote the manuscript with input from all authors.\u003c/p\u003e\u003cp\u003eH.D.B. and O.N. supervised the research.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e\u003cp\u003eWe thank Dr. Y. Goto and Dr. H. Suga for providing the chemically synthesized Asp-DBE substrate. Cryo-EM data were collected at the Cryo-EM facility of The University of Tokyo.\u003c/p\u003e\u003cp\u003eThis work was supported by the \u0026ldquo;N-FLAMS\u0026rdquo; project from the Agence Nationale de la Recherche (ANR-20-CE44-0002), and by the Integrative Molecular and Cellular Biology (IMCBio) program as part of the Interdisciplinary Thematic Institutes (ITI) 2021\u0026ndash;2028 initiative of the University of Strasbourg, CNRS, and INSERM, supported by IdEx Unistra (ANR-10-IDEX-0002) and EUR IMCBio (ANR-17-EURE-0023), under the framework of the French Investments for the Future Program (to N.Y., N.M., S.Z., B.S., F.F., and H.D.B.).\u003c/p\u003e\u003cp\u003eAdditional support was provided by the University of Strasbourg and CNRS (to N.Y., N.M., S.Z., B.S., F.F., and H.D.B.), by JSPS KAKENHI Grant Number 23KJ0722 (to H.M.), and by JST CREST JPMJCR20E2 (to O.N.).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eRoy H, Ibba M (2008) RNA-dependent lipid remodeling by bacterial multiple peptide resistance factors. \u003cem\u003eProc. 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Nat Methods 3:357\u0026ndash;359\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKatoh T, Suga H (2019) Flexizyme-catalyzed synthesis of 3\u0026prime;-aminoacyl-NH-tRNAs. Nucleic Acids Res 47\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePerham RN, SWINGING ARMS AND SWINGING DOMAINS IN (2000) MULTIFUNCTIONAL ENZYMES: Catalytic Machines for Multistep Reactions. www.annualreviews.org\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eNureki O et al (1998) Enzyme Structure with Two Catalytic Sites for Double-Sieve Selection of Substrate. Sci (1979) 280:578\u0026ndash;582\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eFukai S et al (2000) Structural Basis for Double-Sieve Discrimination of L-Valine from L-Isoleucine and L-Threonine by the Complex of tRNAVal and Valyl-tRNA Synthetase. Cell 103:793\u0026ndash;803\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWeete JD, Abril M, Blackwell M (2010) Phylogenetic Distribution of Fungal Sterols. PLoS ONE 5:e10899\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Methods","content":"\u003ch2\u003ePreparation of ErdS for Cryo-EM\u003c/h2\u003e\n\u003cp\u003eThe gene encoding \u003cem\u003eA. fumigatus \u003c/em\u003eErdS\u003csub\u003e\u0026Delta;\u003c/sub\u003e\u003csub\u003e85\u003c/sub\u003e (residues 86-947) was cloned into a pE-SUMO vector. The N-terminally His\u003csub\u003e6\u003c/sub\u003e-SUMO-tagged ErdSDN85 was expressed in \u003cem\u003eEscherichia coli\u003c/em\u003e\u0026nbsp;Rosetta2 (DE3). The\u0026nbsp;\u003cem\u003eE. coli\u003c/em\u003e\u0026nbsp;cells were cultured at 37\u0026thinsp;\u0026deg;C until the\u0026nbsp;\u003cem\u003eA\u003c/em\u003e\u003csub\u003e600\u003c/sub\u003e\u0026nbsp;reached 0.6, and protein expression was induced by adding 0.2\u0026thinsp;mM isopropyl \u0026beta;-D-thiogalactopyranoside (NacalaiTesque). The\u0026nbsp;\u003cem\u003eE. coli\u003c/em\u003e\u0026nbsp;cells were further cultured at 20\u0026thinsp;\u0026deg;C overnight, collected by centrifugation, resuspended in lysis buffer (20\u0026thinsp;mM Tris-HCl, pH 8.0, 300 mM NaCl, 20 mM imidazole, 50 \u0026micro;M TCEP), added 50 \u0026micro;M PMSF and then lysed by sonication. The lysate was centrifuged, and the supernatant was incubated with Ni-NTA Agarose resin (Qiagen) for 1\u0026thinsp;h. The resin was washed with lysis buffer, and the protein was then eluted with elution buffer (20\u0026thinsp;mM Tris-HCl, pH 8.0, 300 mM NaCl, 300 mM imidazole, 50 \u0026micro;M TCEP). The eluted protein was treated with the SUMO protease and dialyzed against dialysis buffer (20\u0026thinsp;mM Tris-HCl, pH 8.0, 50 mM NaCl, 40 mM imidazole, 50 \u0026micro;M TCEP). The protein sample was passed through a Ni-NTA Agarose column, to remove the His\u003csub\u003e6\u003c/sub\u003e-SUMO and SUMO protease. The protein was then loaded onto a 5\u0026nbsp;mL HiTrap SP HP column (GE Healthcare), equilibrated with buffer (20\u0026thinsp;mM Tris-HCl, 50 mM NaCl, 50 \u0026micro;M TCEP).\u0026nbsp;The protein was further purified by chromatography on a HiLoad 16/600 Superdex 200 column (GE Healthcare), equilibrated in elution buffer (20\u0026thinsp;mM Tris-HCl, pH 8.0, 150 mM NaCl, 5 mM MgCl\u003csub\u003e2\u003c/sub\u003e, 50 \u0026micro;M TCEP). The purified ErdS\u0026Delta;85 was concentrated to 2 mg/mL by ultrafiltration, frozen in liquid nitrogen, and stored at \u0026minus;80\u0026deg;C.\u003c/p\u003e\n\u003cp\u003eThe full-length \u003cem\u003eA. fumigatus \u003c/em\u003eErdS (residues 1-947) was also expressed and purified in the same way as ErdSD85. Except that a 50 mM NaCl concentration was used as a buffer and an affinity chromatography column, HiTrap Heparin (GE Healthcare), was used instead of the HiTrap SP HP used during purification. The purified ErdS was concentrated to 3 mg/mL by ultrafiltration, frozen in liquid nitrogen, and stored at \u0026minus;80\u0026deg;C.\u003c/p\u003e\n\u003cp\u003eErdS variant harboring nine substitutions at the AspRS interaction interfacewas also expressed and purified in the same way as full-length ErdS. Based on our ErdS structure, we identified nine residues in AspRS domain that interact with the tRNA\u003csup\u003eAsp\u003c/sup\u003e acceptor stem. To weaken this interaction, these residues were mutated as follows: Q334E, S335A, N362P, S363A, N364P, H366A, R367A, H368A, and Y494F. The purified ErdS was concentrated to 3 mg/mL by ultrafiltration, frozen in liquid nitrogen, and stored at \u0026minus;80\u0026deg;C.\u003c/p\u003e\n\u003ch2\u003ePreparation of tRNA\u003csup\u003eAsp\u003c/sup\u003e and Asp-\u003cem\u003eN\u003c/em\u003e-tRNA\u003csup\u003eAsp\u003c/sup\u003e for Cryo-EM\u003c/h2\u003e\n\u003cp\u003eThe gene encoding \u003cem\u003eA. fumigatus \u003c/em\u003etRNA\u003csup\u003eAsp\u003c/sup\u003e in which a hammerhead ribozyme sequence and the consensus T7 promoter sequenceon its 5\u0026prime; end was cloned into a pUC119 vector. tRNA\u003csup\u003eAsp\u003c/sup\u003e was transcribed in vitro at 37\u0026deg;C for 16-20 h using T7 polymerase, followed by incubation at 60\u0026deg;C for 16-20 h with the addition of MgCl\u003csub\u003e2\u003c/sub\u003e to a final concentration of 30 mM. The target tRNA bands were separated by urea-PAGE, extracted by EluTrap, ethanol precipitated, buffer exchanged, and stored at \u0026minus;80\u0026deg;C.\u003c/p\u003e\n\u003cp\u003eFor the preparation of the Asp-\u003cem\u003eN\u003c/em\u003e-tRNA\u003csup\u003eAsp\u003c/sup\u003e, 3\u0026prime;-Overlapping forward and reverse DNA oligonucleotides (Integrated DNA Technologies) were hybridized and enzymatically extended to form a dsDNA template in which a hammerhead ribozyme sequence was sandwiched between the consensus T7 promoter sequence and the coding sequence for \u003cem\u003eAfm\u003c/em\u003etRNA\u003csup\u003eAsp\u003c/sup\u003e as described\u003csup\u003e1\u003c/sup\u003e. To suppress non-templated transcription, the 5\u0026prime;-end of the reverse oligo contained two 2\u0026prime;-OMe nucleotides. tRNA\u003csup\u003eAsp\u003c/sup\u003e was transcribed in vitro at 37\u0026deg;C for 6 h using T7 polymerase, followed by incubation at 60-65 \u0026deg;C for 1 h with the addition of MgCl\u003csub\u003e2\u003c/sub\u003e to a final concentration of 30 mM. The target tRNA bands were separated by urea-PAGE, crushed with a glass rod, and extracted into TE by shaking overnight. After centrifugation the supernatant was passed through a 0.45 \u0026micro;m syringe filter (33 mm; Thermo Fisher) and the tRNA was ethanol precipitated.\u003c/p\u003e\n\u003cp\u003e3\u0026prime;-amino-tailing followed the protocol described in\u003csup\u003e2\u003c/sup\u003e. Briefly, reactions containing 100 \u0026micro;M tRNA\u003csup\u003eAsp\u003c/sup\u003e, 32 \u0026micro;M human CCA-adding enzyme,1.75 mM 3\u0026prime;-amino-ATP (BioLog Life Science Institute, Bremen, GER), 1 mM sodium pyrophosphate, 10 mM MgCl\u003csub\u003e2\u003c/sub\u003e, 1.0 mM DTT, 0.2 units/\u0026micro;L murine RNase inhibitor (NEB), and 50 mM glycine (pH 9.0) were incubated for 1 hr at 37\u0026deg;C. Prior to workup pyrophosphate was hydrolyzed by adding inorganic pyrophosphatase (0.1 U/\u0026micro;l; New England Biolabs) to the reaction and incubating an additional 15 min at RT. Each reaction was quenched with 2.5 M NaOAc (pH 5.0), extracted with an equal volume of pH 5.0 phenol:chloroform-isoamyl alcohol (80:17:3), and the tRNA ethanol precipitated. The extent of 3\u0026prime;-tailing was determined to be 60% by biotin-streptavidin gel shift assay\u003csup\u003e3\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eAmino-tailed tRNA was stably charged by incubation with flexizyme (dFx) and Asp-DBE\u003csup\u003e4,5\u003c/sup\u003e. Briefly, reactions containing 40 \u0026micro;M tRNA (60% amino-tailed), 31 \u0026micro;M flexizyme, 3 mM Asp-DBE, 375 mM MgCl\u003csub\u003e2\u003c/sub\u003e, 12.5% DMSO, and 0.1 M K-HEPES (pH 7.5) were incubated in a thermomixer at 25 \u0026deg;C with shaking for 72 hr. After 24 and 48 hr each reaction was supplemented with fresh 200 mM Asp-DBE. Reactions were quenched with 0.1 volume 2.5 M NaOAc pH 5.0 followed by ethanol precipitation. The combined pellets were dissolved in 0.1 M glycine pH 9.0 and incubated 3 hrs at 37 \u0026deg;C to hydrolyze alkali-labile aminoacyl ester linkages. After ethanol precipitation tRNA was purified away from flexizyme by urea-PAGE. Since stably charged tRNA could not be resolved from amino-tailed tRNA by gel electrophoresis, mass spectrometry was used to determine the charging efficiency, which proved to be 40%.\u003c/p\u003e\n\u003ch2\u003eCryo-EM sample preparation,data collection and image processing\u003c/h2\u003e\n\u003cp\u003eFor the ErdS tetramer structure, ErdS\u003csub\u003e\u0026Delta;85\u003c/sub\u003e and tRNA\u003csup\u003eAsp\u003c/sup\u003e were mixed at a molar ratio of 1:1 and incubated at 37\u0026deg;C for 20 min. The 3 \u0026micro;L of the prepared sample was applied to a freshly glow-discharged Cu/Rh 300 mesh R1.2/1.3 grid (Quantifoil), in a Vitrobot Mark IV (FEI) at 4\u0026thinsp;\u0026deg;C, with a waiting time of 30\u0026thinsp;s and a blotting time of 4\u0026thinsp;s under 100% humidity conditions.\u0026nbsp;The grid was plunge-frozen in liquid ethane cooled at liquid nitrogen temperature.\u003c/p\u003e\n\u003cp\u003eCryo-EM data were collected using a Titan Krios G3i microscope (Thermo Fisher Scientific), running at 300\u0026thinsp;kV and equipped with a Gatan Quantum-LS Energy Filter (GIF) and a Gatan K3 Summit direct electron detector in the electron counting mode. Micrographs were recorded at a nominal magnification of 105,000\u0026times;, corresponding to a calibrated pixel size of 0.83\u0026Aring;, with a total dose of 49 electrons per \u0026Aring;\u003csup\u003e2\u003c/sup\u003e. The data were automatically collected by the image shift method using the SerialEM software, with a defocus range of \u0026minus;1.6 to \u0026minus;0.8\u0026thinsp;\u0026mu;m, and 3,357 movies were obtained and processed using RELION-3.1\u003csup\u003e6\u0026ndash;10\u003c/sup\u003e. From the 3,357 motion-corrected and dose-weighted micrographs, 2,021,179 particles were initially picked, and extracted at a pixel size of 3.32\u0026thinsp;\u0026Aring;. These particles were subjected to several rounds of 2D and 3D classifications. The selected 240,624 particles were re-extracted at a pixel size of 1.66\u0026thinsp;\u0026Aring;, and then subjected to 3D refinement, per-particle defocus refinement, beam-tilt refinement, Bayesian polishing, and 3D classification with the mask focusing on ErdS monomer\u003csup\u003e11\u003c/sup\u003e. Finally, a single class of 35,812particles was selected after signal subtraction and symmetry expansion to \u003cem\u003eC\u003c/em\u003e\u003csub\u003e1\u003c/sub\u003e, yielding a map at 3.6\u0026thinsp;\u0026Aring; resolution, according to the Fourier shell correlation (FSC)\u0026thinsp;=\u0026thinsp;0.143 criterion\u003csup\u003e12\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eAs for the ErdS/tRNA\u003csup\u003eAsp\u003c/sup\u003e complex, the tRNA\u003csup\u003eAsp\u003c/sup\u003e that had been aspartylated by AspRS was mixed with twice the molar volume of ErdS and ergosterol to a final concentration of 630 \u0026micro;M were incubated at room temperature for 1 h. The sample was added to a previously glow discharged 300 mesh Au R1.2/1.3 grid (Quantifoil) for cryo-EM observation and rapidly frozen.\u003c/p\u003e\n\u003cp\u003eCryo-EM data were collected using a Titan Krios G4 microscope (Thermo Fisher Scientific), running at 300\u0026thinsp;kV and equipped with a Gatan Quantum-LS Energy Filter (GIF) and a Gatan K3 Summit direct electron detector in the electron counting mode. Micrographs were recorded at a nominal magnification of 105,000\u0026times;, corresponding to a calibrated pixel size of 0.83\u0026Aring;, with a total dose of 48 electrons per \u0026Aring;\u003csup\u003e2\u003c/sup\u003e. The data were automatically collected by the image shift method using the EPU software (Thermo Fisher Scientific), with a defocus range of \u0026minus;1.6 to \u0026minus;0.4\u0026thinsp;\u0026mu;m, and 2,334 movies were obtained and processed using cryoSPARC v4\u003csup\u003e13\u003c/sup\u003e. Initial particle picks were used as templates for repicking using Topaz, yielding 885,089 particles from all micrographs, and extracted\u003csup\u003e14\u003c/sup\u003e. These particles were subjected to 2D classification, and the selected 510,043 particles were combined for multiple rounds of three-dimensional classification (ab initio model generation and heterogeneous refinement). The best class containing 20,590 particles was refined using non-uniform refinement\u0026nbsp;after CTF refinement, yielding a map at 3.66\u0026thinsp;\u0026Aring; resolution\u003csup\u003e15\u003c/sup\u003e.To see the ATT domain, particles were subjected to several rounds of three-dimensional classification (3D refinement and heterogenous refinement). The best class containing 15,808 particles was refined using Non-uniform Refinement, yielding a map at 3.43\u0026thinsp;\u0026Aring; resolution.\u003c/p\u003e\n\u003cp\u003eAs for the ErdS/tRNA\u003csup\u003eAsp\u003c/sup\u003e complex in tRNA moved state,the Asp-N-tRNA\u003csup\u003eAsp\u003c/sup\u003e was mixed with ErdS variant at a molar ratio of 0.2:1 and incubatedat room temperature for 1 h. The sample was added to a previously glow discharged 300 mesh Au R1.2/1.3 grid (Quantifoil) for cryo-EM observation and rapidly frozen.\u003c/p\u003e\n\u003cp\u003eCryo-EM data were collected using a Titan Krios G3i microscope (Thermo Fisher Scientific), running at 300\u0026thinsp;kV and equipped with a Gatan Quantum-LS Energy Filter (GIF) and a Gatan K3 Summit direct electron detector in the electron counting mode. Micrographs were recorded at a nominal magnification of 105,000\u0026times;, corresponding to a calibrated pixel size of 0.83\u0026Aring;, with a total dose of 51 electrons per \u0026Aring;\u003csup\u003e2\u003c/sup\u003e. The data were automatically collected by the image shift method using the EPU software (Thermo Fisher Scientific), with a defocus range of \u0026minus;1.6 to \u0026minus;0.4\u0026thinsp;\u0026mu;m, and 7,809 movies were obtained and processed using cryoSPARC v4. Initial particle picks were used as templates for repicking using Topaz, yielding 3,038,832 particles from all micrographs, and extracted. These particles were subjected to 2D classification, and the selected 354,720 particles were combined for multiple rounds of three-dimensional classification (ab initio model generation and 3D refinement). The best class containing 34,200 particles was refined using non-uniform refinement\u0026nbsp;after CTF refinement, yielding a map at 3.65\u0026thinsp;\u0026Aring; resolution.\u003c/p\u003e\n\u003ch2\u003eModel building, validation and superimpositions\u003c/h2\u003e\n\u003cp\u003eFor atomic model building, initialErdS models were generated by AlphaFold2\u003csup\u003e16\u003c/sup\u003e. The initial tRNAAsp was derived from the crystal structure of yeast tRNA\u003csup\u003eAsp\u003c/sup\u003e (PDB 1ASY)17. The models were manually docked into cryo-EM map and modeled in Coot andrefined using phenix.real_space_refine\u003csup\u003e17,18\u003c/sup\u003e. The structure validation was performed using MolProbity in the PHENIX package\u003csup\u003e19\u003c/sup\u003e. The statistics of the 3D reconstruction and model refinement are summarized in Extended Data Table 1. The cryo-EM density map figures and molecular graphics were generated using UCSF ChimeraX\u003csup\u003e20,21\u003c/sup\u003e. Models were superimposed using the match maker in ChimeraX. Ergosterol was positioned and relaxed by Sphere Refine on COOT.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e Fungal strains and growth media\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll strains and derivatives used in this study are listed in \u003cstrong\u003eTable S1\u003c/strong\u003e.\u003cem\u003eS. cerevisiae \u003c/em\u003estrains used for plasmid shuffling are also presented in \u003cstrong\u003eTable S2\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eS. cerevisiae \u003c/em\u003estrains BY4742 (\u003cem\u003eMAT\u0026alpha; his3\u0026Delta;1 leu2\u0026Delta;0 lys2\u0026Delta;0 ura3\u0026Delta;0\u003c/em\u003e) were used to express WT ErdS and mutants from pRS415 (LEU) plasmids. They were routinely grown on SC-Leu agar (MP\u0026trade; Biomedicals) at 30 \u0026deg;C. The \u003cem\u003eS. cerevisiae \u003c/em\u003eYAL3 (\u003cem\u003edps1\u003c/em\u003eD) shuffle strain (\u003cem\u003eMATa ura3-52 lys2-801am trp1-63 his3-200 leu2-1 ade2-450 ade3-1483 dps1\u003c/em\u003e::\u003cem\u003eHIS3\u003c/em\u003e) contained a rescue plasmid (TRP) and pRS415 (LEU) plasmids carrying WT of mutant versions of ErdS. Before shuffling they were grown on SC-Leu-Trp at 30 \u0026deg;C, and after shuffling, when the rescue plasmid was chased, on SC-Leu agar (MP\u0026trade; Biomedicals). For liquid cultures, the same media without agar were used in glass flasks, cultures incubated at 30 \u0026deg;C under shaking (200 rpm).\u003c/p\u003e\n\u003cp\u003eFor \u003cem\u003eAspergillus fumigatus\u003c/em\u003e, the CEA17 D\u003cem\u003eakuB\u003c/em\u003e\u003csup\u003eKU80\u003c/sup\u003e strain (\u003cem\u003eaka\u003c/em\u003e \u0026ldquo;WT Ku80\u0026rdquo;) and derivatives were used \u003cem\u003eA. fumigatus\u003c/em\u003e was maintained as follows: fresh conidia were spread on Malt agar (Thermo Scientific) plates or slants and incubated in the dark at 37 \u0026deg;C until it produced enough mature conidia. For phenotypic characterization, strains were grown on Malt agar or on Minimal Medium (MM) Agar plates (For 1 L, MM contained glucose (2 % w/v) or xylose (2 % w/v), ammonium tartrate dibasic (0.92 g, 5 mM, \u003cem\u003ei.e.\u003c/em\u003e, 10 mM ammonium), salts (10 mL of a 50 X solution containing KCl 26 g/L, MgSO\u003csub\u003e4\u003c/sub\u003e 7H2O 26 g/L, KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e 76 g/L) and trace elements (0.5 mL of a 1000 X solution containing: FeSO\u003csub\u003e4\u003c/sub\u003e 7H\u003csub\u003e2\u003c/sub\u003eO 1 g, Na\u003csub\u003e2\u003c/sub\u003eEDTA 10 g, ZnSO\u003csub\u003e4\u003c/sub\u003e 7H\u003csub\u003e2\u003c/sub\u003eO 4.4 g, H\u003csub\u003e3\u003c/sub\u003eBO\u003csub\u003e3 \u003c/sub\u003e2.2 g, MnCl\u003csub\u003e2\u003c/sub\u003e 4H\u003csub\u003e2\u003c/sub\u003eO 1 g, CoCl\u003csub\u003e2 \u003c/sub\u003e6H\u003csub\u003e2\u003c/sub\u003eO 0.32 g, CuSO\u003csub\u003e4 \u003c/sub\u003e5H\u003csub\u003e2\u003c/sub\u003eO 0.32 g and Na\u003csub\u003e2\u003c/sub\u003eMoO\u003csub\u003e4\u003c/sub\u003e0.8 g for 200 mL adjusted at pH 6.5 ; 1.5 % (w/v) agar was added). For liquid cultures, \u003cem\u003eAfm\u003c/em\u003e was grown in liquid MM with 1 % (w/v) glucose. Liquid cultures were performed in glass flasks at 37 \u0026deg;C (\u003cem\u003eAfm\u003c/em\u003e) under shaking (200 rpm) for 24 h (\u003cem\u003eAfm\u003c/em\u003e, \u003cem\u003eSce\u003c/em\u003e) or 48 h (\u003cem\u003eNcr\u003c/em\u003e). For \u003cem\u003eNeurospora crassa\u003c/em\u003e, the 74-OR23-1VA WT (Fungal Genetic Stock Center, strain FGSC#2489) and D\u003cem\u003eerdH\u003c/em\u003e (FGSC#20235) strains were employed. \u003cem\u003eN. crassa \u003c/em\u003ewas grown on nutrient rich plates (Glucose 4 % w/v, Peptone 1 % w/v and Yeast extract 2 % w/v), or in MM liquid medium at 30 \u0026deg;C. \u003cem\u003eMagnaporthe oryzae\u003c/em\u003e strains (isolate Guy11) were routinely grown, maintained and stored as previously described\u003csup\u003e22\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConstruction of \u003cem\u003eA. fumigatus \u003c/em\u003estrains\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConstruction of the \u003cem\u003eAfm\u003c/em\u003eD\u003cem\u003eerdS\u003c/em\u003e and D\u003cem\u003eerdS\u003c/em\u003e::P\u003cem\u003e\u003csub\u003eXyl\u003c/sub\u003e\u003c/em\u003e-\u003cem\u003eerdS\u003c/em\u003e strains was described in\u003csup\u003e23\u003c/sup\u003e. The WT::P\u003cem\u003e\u003csub\u003eXyl\u003c/sub\u003e\u003c/em\u003e-\u003cem\u003eerdS\u003c/em\u003e strain was constructed using the same integration cassette as that used for the D\u003cem\u003eerdS\u003c/em\u003e::P\u003cem\u003e\u003csub\u003eXyl\u003c/sub\u003e\u003c/em\u003e-\u003cem\u003eerdS\u003c/em\u003e strain. It was designed from the \u003cem\u003esix-\u003c/em\u003eP\u003cem\u003e\u003csub\u003exyl\u003c/sub\u003e\u003c/em\u003e-b\u003cem\u003erec\u003c/em\u003e-\u003cem\u003etrpC\u003c/em\u003e-\u003cem\u003ehygB\u003c/em\u003e-\u003cem\u003esix \u003c/em\u003eresistance cassette (with the b-recombinase \u003cem\u003esix\u003c/em\u003e sequences)\u003csup\u003e24\u003c/sup\u003e surrounded by the 5\u0026rsquo; and 3\u0026rsquo; 1000-bp flanking regions of the \u003cem\u003eerdS\u003c/em\u003e gene.The \u003cem\u003eerdS\u003c/em\u003e gene was inserted in place of the b-recombinase gene between the P\u003cem\u003e\u003csub\u003eXyl\u003c/sub\u003e\u003c/em\u003e promoter and TrpC terminator\u003csup\u003e23\u003c/sup\u003e.Transformations of \u003cem\u003eA. fumigatus \u003c/em\u003eCEA17 D\u003cem\u003eakuB\u003c/em\u003e\u003csup\u003eKU80 \u003c/sup\u003ewas performed by electroporation of swollen conidia essentially as described\u003csup\u003e25\u003c/sup\u003e and we used 10 \u0026mu;g of linearized (\u003cem\u003eSma\u003c/em\u003eI-digested) plasmid containing the replacement cassette. From the primary hygromycin-resistant colonies, conidia were isolated on individual Malt agar plates containing 150 \u0026mu;g/mL hygromycin B. After 3 days, conidia from single isolated colonies were scrapped and transferred on Malt agar slants containing 150 \u0026mu;g/mL hygromycin B and incubated 5 days at 37 single iso DNA was extracted\u003csup\u003e26\u003c/sup\u003efrom mycelium cultures ofmutants and replacement of the\u003cem\u003eAfm-erdS\u003c/em\u003egene was confirmed by PCR (Primers FF#684+NY#015 and NY#016+FF#685).\u003c/p\u003e\n\u003cp\u003eConstruction of \u003cem\u003eAfm\u003c/em\u003e strains expressing ErdS-eGFP and ErdH-mCherry. With the E-Zyvec company (now Polyplus), we constructed plasmids containing the \u003cem\u003eerdH\u0026mdash;erdS\u003c/em\u003elocus found in \u003cem\u003eAfm\u003c/em\u003e that we modified by fusion codon-optimized ORFs of eGFP and mCherry, respectiverly at the 3\u0026rsquo;-end of \u003cem\u003eerdS\u003c/em\u003e and \u003cem\u003eerdH\u003c/em\u003e. Both genes were thus under the control of their own natural promoter (P\u003cem\u003e\u003csub\u003eerd\u003c/sub\u003e\u003c/em\u003e). This construct was inserted before the \u003cem\u003esix-\u003c/em\u003eP\u003cem\u003e\u003csub\u003exyl\u003c/sub\u003e\u003c/em\u003e-\u0026beta;\u003cem\u003erec\u003c/em\u003e-\u003cem\u003etrpC\u003c/em\u003e-\u003cem\u003ehygB\u003c/em\u003e-\u003cem\u003esix\u003c/em\u003e cassette. The 5\u0026rsquo;- and 3\u0026rsquo;-flanking regions of the \u003cem\u003eerdH\u0026mdash;erdS\u003c/em\u003elocus were added, to obtain the 5\u0026rsquo;-flank_\u003cem\u003eerdH-mCherry\u0026mdash;erdS-eGFP-six-\u003c/em\u003eP\u003cem\u003e\u003csub\u003exyl\u003c/sub\u003e\u003c/em\u003e-\u0026beta;\u003cem\u003erec\u003c/em\u003e-\u003cem\u003etrpC\u003c/em\u003e-\u003cem\u003ehygB\u003c/em\u003e-\u003cem\u003esix_\u003c/em\u003e3\u0026rsquo;_flankcassette, surrounded by SmaI restriction sites. The resulting plasmid was digested using SmaI and transformed in the \u003cem\u003eA. fumigatus \u003c/em\u003eCEA17 D\u003cem\u003eakuB\u003c/em\u003e\u003csup\u003eKU80\u003c/sup\u003e(WT Ku80) strain by electroporation, as described. Hygromycin-resistant colonies were selected on MM medium (pH 6.8) containing 150 \u0026micro;g/mL hygromycin B and purified through two successive re-streaks on Malt agar containing 150 \u0026micro;g/mL hygromycin. Strains that presented red and green fluorescence were then grown on MM containing 2 % (w/v) xylose to excise the \u003cem\u003esix-\u003c/em\u003eP\u003cem\u003e\u003csub\u003exyl\u003c/sub\u003e\u003c/em\u003e-\u0026beta;\u003cem\u003erec\u003c/em\u003e-\u003cem\u003etrpC\u003c/em\u003e-\u003cem\u003ehygB\u003c/em\u003e-\u003cem\u003esix\u003c/em\u003e cassette, and to ensure that only the 5\u0026rsquo;-flank_\u003cem\u003eerdH-mCherry\u0026mdash;erdS-eGFP-_\u003c/em\u003e3\u0026rsquo;_flank construct remained. Two independent clones were selected for epifluorescence observations. Strains were verified by PCR.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConstruction of \u003cem\u003eM. oryzae \u003c/em\u003estrains (CrispR)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe \u003cem\u003eerdS\u003c/em\u003e (MGG_13783) and \u003cem\u003eerdH\u003c/em\u003e (MGG_04680) genomic sequences were retrieved from EnsemblFungi(https://fungi.ensembl.org/). \u003cem\u003eerdS\u003c/em\u003e and \u003cem\u003eerdH\u003c/em\u003e were deleted using the RNP-mediated CrispR editing strategy as previouslyadapted in \u003cem\u003eM. oryzae\u003csup\u003e27\u003c/sup\u003e\u003c/em\u003e. To generate the donor DNAs, the BAR (glufosinate-ammonium resistance) cassette was amplified from the pCB1530 plasmid (FGSC) using approx. 100 bplong primers leading to amplicons containing the BAR cassette flanked by \u003cem\u003eerdS\u003c/em\u003e or \u003cem\u003eerdH\u003c/em\u003e homologous arms. The sgRNA were obtained by in vitro transcription using the EnGen\u0026reg; sgRNA Synthesis Kit (Neb #3322) following the manufacturer\u0026rsquo;s instructions. All primers used to generate and verify the strains are listed in \u003cstrong\u003eTable S3\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePlasmids construction\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe \u003cem\u003eAfm erdS \u003c/em\u003eopen reading frame (\u003cem\u003eAFUA_1g02570\u003c/em\u003e) was synthesized (Genscript\u0026reg;) withcodon optimization for expression in \u003cem\u003eSce\u003c/em\u003e, amplified by PCR and cloned in a pRS415 (LEU) yeast expression vector as described in\u003csup\u003e23\u003c/sup\u003e. The p415-\u003cem\u003eAor-erdS\u003c/em\u003e plasmid containing the \u003cem\u003eerdS\u003c/em\u003e gene from \u003cem\u003eAor\u003c/em\u003ewas constructed previously\u003csup\u003e23\u003c/sup\u003e. All ErdS mutants were obtained from these pRS415 plasmids through mutagenesis. Briefly, for site-directed mutagenesis, we proceeded as follows. A pair of overlapping primers containing the desired mutation were used (\u003cstrong\u003eTable S3\u003c/strong\u003e). The \u0026ldquo;reverse\u0026rdquo; primer was used together with a \u0026ldquo;forward\u0026rdquo; primer matching the ampicilline resistance gene of the plasmid (FF#060) to amplify by PCR a first half of the pRS415-\u003cem\u003eerdS\u003c/em\u003e plasmid, containing the desired mutation. The \u0026ldquo;forward\u0026rdquo; primer was used with the \u0026ldquo;reverse\u0026rdquo; primer (FF#061) matching the ampicilline resistance gene of the plasmid to amplify the second half of the plasmid, with the desired region mutated. Then, 1 \u0026micro;L of each PCR reaction were used in a Gibson Assembly reaction mixture to reunite the two plasmid halves. Then, 8 \u0026micro;L of the reaction mixture was used to transform XL-1 Blue chimiocompetent \u003cem\u003eE. coli\u003c/em\u003e cellsto select plasmids in which the ampicilline resistance gene was properly reconstituted. The same strategy was used to obtain the \u003cem\u003eerdS\u003c/em\u003eD(1-90) mutant of \u003cem\u003eAor erdS \u003c/em\u003efrom the pRS415-\u003cem\u003eAor erdS \u003c/em\u003eplasmid. The presence of the desired mutations in \u003cem\u003eerdS\u003c/em\u003e was verified and validated by sequencing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e Plasmid-shuffling complementation assays in \u003cem\u003eS. cerevisiae\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePlasmid shuffling experiments were conducted in the YAL3D\u003cem\u003edps1 Sce\u003c/em\u003estrain\u003csup\u003e28\u003c/sup\u003erescued with awild-type \u003cem\u003eDPS1 \u003c/em\u003egene copy cloned in an \u003cem\u003eURA3\u003c/em\u003e-bearing plasmid. All \u003cem\u003eAfm\u003c/em\u003eor \u003cem\u003eAorerdS\u003c/em\u003econstructsto be tested were cloned in pRS415 (LEU) plasmids, transformed in the D\u003cem\u003edps1 \u003c/em\u003estrain, and shuffledessentially as described\u003csup\u003e28,29\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e Preparation of spores/conidia from \u003cem\u003eA. fumigatus \u003c/em\u003eand \u003cem\u003eN. crassa\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSpores from 7 days-old Malt agar slants or plates were resuspended by addition of 5 mL sterile Tween 20-H\u003csub\u003e2\u003c/sub\u003eO (0.05 % v/v) and vortexing, then the conidia were filtered with Cell Strainer filters (EASY strainer\u003csup\u003eTM\u003c/sup\u003eGreiner Bio-One), and the concentration was determined with a LUNA-FL cell counter (ref.59506, Dutscher). Conidia were stored in Tween 20-H\u003csub\u003e2\u003c/sub\u003eO (0.05 % v/v) at 4 \u0026deg;C for up to 2 week. For \u003cem\u003eNcr\u003c/em\u003e, conidia were harvested from 14 days-old Malt agar slants and treated similarly using 1 M sterile sorbitol.For \u003cem\u003eM. oryzae\u003c/em\u003e, spores were harvested from 7-9 days old cultures grown on CM agar plates. 4 mL were poured on the Petri dish, and spores were released by scratching the mycelia surface using a sterile disposable plastic spreader. The spore solution was then filtered through Miracloth, centrifuged at 3000 x g for 5 min and finally resuspended in 1 mL of water. Cells were counted using a Bright-Line\u0026trade; Hemacytometer (Merck #Z359629).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePhenotypic characterization of \u003cem\u003eA. fumigatus \u003c/em\u003estrains\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor phenotypic characterizations, 10\u003csup\u003e6\u003c/sup\u003e\u003cem\u003eAfm\u003c/em\u003e conidia in 5 \u0026micro;L were point-inoculated on squared agar plates from suspensions prepared as indicated below to obtain individual round-shaped colonies. Each plate was inoculated with the 4 \u003cem\u003eAfm\u003c/em\u003e derivative (WT Ku80, D\u003cem\u003eerdS\u003c/em\u003e, D\u003cem\u003eerdS\u003c/em\u003e::P\u003cem\u003e\u003csub\u003eXyl\u003c/sub\u003e\u003c/em\u003e-\u003cem\u003eerdS\u003c/em\u003e and WT::P\u003cem\u003e\u003csub\u003eXyl\u003c/sub\u003e\u003c/em\u003e-\u003cem\u003eerdS\u003c/em\u003e), as to ensure proper comparison between strains. Colony diameter was measured at indicated time points and colonies were pictured using a cell phone or using an AxioVision magnifier. To produce mycelia lawns, 50 \u0026micro;L of a conidia suspension at 10\u003csup\u003e7\u003c/sup\u003e conidia/mL were spread on MM agar medium-containing wells of 12-well plates. Each plate featured 3 replicates of the 4 strains tested. Lawns were pictured using an AxioVision magnifier. All plates were incubated at 37 \u0026deg;C in the dark and documented at the indicated time points.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e Liquid cultures and mycelia harvesting\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLiquid cultures to produce mycelia for total lipids extraction were inoculated with 10\u003csup\u003e7\u003c/sup\u003e conidia/mL in 50 mL liquid MMG (MM + 1 % (w/v) glucose), incubated for 24 h at 37 \u0026deg;C (\u003cem\u003eAfm\u003c/em\u003e) or 48 h at 30 \u0026deg;C(\u003cem\u003eNcr\u003c/em\u003e) in the dark under agitation (220 rpm). To test the overproduction of Erg-Asp, the 4 \u003cem\u003eAfm\u003c/em\u003e strains were grown in liquid MM + 2 % (w/v) glucose (MMG) or MM + 2 % (w/v) xylose (MMX). Experiments were also conducted with cultures in the presence of various ratios of glucose and xylose. Mycelia were then filtrated through two layers of gauze, rinsed twice with 50 mL sterile H\u003csub\u003e2\u003c/sub\u003eO, and squeezed to eliminate excess water. Mycelia were directly used to extract total lipids.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGermination assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo investigate the role of Erg-Asp in germination process, conidia suspension (1x10\u003csup\u003e6\u003c/sup\u003e conidia /mL) from WT Ku80 and D\u003cem\u003eerdS \u003c/em\u003estrains were prepared. Germination assays were performed in 1 mL MM medium in Eppendorf tubes, incubated at 37 \u0026deg;C with agitation at 200 rpm. Microscopic observation began 3 hours after incubation, then continued hourly. At each time point, samples were collected and centrifuged at maximum speed for 10 min at 4\u0026deg;C. Supernatants were removed, and pellets were fixed in cold 4% paraformaldehyde (4% PFA) for 15 min at room temperature. After a second centrifugation (10 min, max speed, 4\u0026deg;C) and removal of the fixative, conidia were resuspended in 1mL, sterile 1xPBS (phosphate buffered saline) and stored \u0026agrave; 4 \u0026deg;C until microscopy observations. Bright field images were captured hourly using a Zeiss Axiovert 200 epifluorescence microscope with a 65x objective. Germination was manually assessed by examining several fields of view for each strain and each time point. Conidia were outlined manually, totaling approximately 100 fungal cells per strain per time point. Observations were recorded hourly 3 to 8 hours post-incubation. Germination percentages at the germ tube stage were compared between WT Ku80 and \u0026Delta;\u003cem\u003eerdS\u003c/em\u003e strains over time using a two-way ANOVA statistical test performed with GraphPad Prism. Multiple comparisons were conducted using the Sidak test, with a significance threshold set at 0.05.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNanolive microscopy\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eCell Culture.\u003c/em\u003e Murine macrophage IC-21 cells were cultured under standard conditions at 37 \u0026deg;C with 5 % CO\u003csub\u003e2\u003c/sub\u003e. They were maintained in complete growth medium (RPMI+FBS+PS) and seeded for two different experiments in 35 mm ibidi low border dishes. On July 18, 2023, five dishes were seeded with 60,000 cells each, while on July 24, 2023, two additional dishes were seeded with 80,000 cells each. \u003cem\u003eInfection Preparation and Conditions:\u003c/em\u003e For the first experiment (WT Ku80 conidia), one dish was used to assess cell viability following a 30-minute incubation at 4 \u0026deg;C, a condition necessary for synchronizing phagocytosis. A second dish was used for cell counting to calculate the appropriate number of spores needed to reach the desired multiplicity of infection (MOI). The remaining three dishes were used for infection with WT Ku80 spores: one dish served as a negative control, one was infected at MOI 5, and the third at MOI 10. To achieve this, the stock solution (2.36 \u0026times; 10⁹ spores in 4 mL) was first pre-diluted 1:200 (3 \u0026micro;L stock + 597 \u0026micro;L medium) to obtain 1.77 \u0026times; 10⁶ spores in 600 \u0026micro;L. For an MOI of 10, 237 \u0026micro;L of this pre-dilution (corresponding to 700,000 spores) was added to 70,000 IC-21 cells in a final volume of 800 \u0026micro;L. For an MOI of 5, 119 \u0026micro;L (350,000 spores) was used under the same conditions.After 24 h of incubation, spores were added to the respective dishes. To synchronize phagocytosis, all infected dishes were placed at 4 \u0026deg;C for 30 min, followed by a medium change to remove unbound spores.In the second experiment (\u003cem\u003e∆erdS\u003c/em\u003e spores) realized on July 24, 2023, IC-21 cells were infected with \u003cem\u003e∆erdS\u003c/em\u003e spores at MOI 5 and MOI 10. In this case, the stock solution (3.28 \u0026times; 10⁹ spores in 4 mL) was also pre-diluted 1:200 (3 \u0026micro;L stock + 597 \u0026micro;L medium) to obtain 2.46 \u0026times; 10⁶ spores in 600 \u0026micro;L. For an MOI of 10, 196 \u0026micro;L of this pre-dilution (corresponding to 800,000 spores) was added to 80,000 IC-21 cells per dish in a final volume of 800 \u0026micro;L. For an MOI of 5, 98 \u0026micro;L (400,000 spores) was used under the same conditions. The spore solution was taken out of the fridge for 15 min before dilution to reach room temperature and the cells were taken out of the incubator 5 min before adding the spores. As in the previous experiment, cells were incubated with spores at 4 \u0026deg;C for 30 min, then washed by changing the medium to eliminate unbound spores.All \u003cem\u003eAfm\u003c/em\u003e spore preparations were performed one by one with complete disinfection in between to avoid any risk of cross-contamination between spore types. \u003cem\u003eImage Acquisition:\u003c/em\u003e Following spore addition and washing, live-cell imaging was performed using a CX96-Focus (Nanolive SA, Tolochenaz, Switzerland) microscope. The microscope is equipped for long-term live cell imaging: temperature, humidity, and gas composition. The incubator chamber (Okolab) keeps the sample at 37 \u0026deg;C, is closed by a heating glass lid to prevent condensation and is connected to a gas mixer (2GF-Mixer, Okolab) to maintain 5% of CO\u003csub\u003e2\u003c/sub\u003e. The humidity module ensures a 90% relative humidity within the chamber. The first experiment was done with a 3\u0026times;3 grid scan with one image every 2min9s, while the second used a 4\u0026times;4 grid scan with one image every 2min21s.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e Fluorescence microscopy of \u003cem\u003eA. fumigatus\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConidia from two independent\u003cem\u003eAfm\u003c/em\u003e strains carrying the \u003cem\u003eerdH-mCherry\u0026mdash;erdS-eGFP\u003c/em\u003e locus were used to inoculate liquid YG medium (1 % Yeast extract, 2 % glucose) (10\u003csup\u003e5\u003c/sup\u003e conidia/mL). Development of the fungus was followed at the indicated time points. At each time point, 1 mL of culture was aliquoted, conidia or mycelia centrifugated and washed with phosphate buffer saline (PBS), resuspended in PBS and 10 \u0026micro;L of resuspended strains were used to prepare slides. Epifluorescence imageswere taken with an AXIO Observer d1 (Carl Zeiss) epifluorescence microscope using a 100 \u0026times; plan apochromatic objective (Carl Zeiss) and processed with the Image J software.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e Total lipids extraction\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOvernight \u003cem\u003eSce\u003c/em\u003ecultures were diluted to an OD\u003csub\u003e600\u003c/sub\u003e = 0.1, grown (220 rpm) until the OD\u003csub\u003e600\u003c/sub\u003e was ~1 and cells were harvested by centrifugation at 5000\u0026times;\u003cem\u003eg\u003c/em\u003e for 15 min at 4\u0026deg;C. \u003cem\u003eAfm\u003c/em\u003eor \u003cem\u003eNcr\u003c/em\u003econidia were harvested as described and inoculated in glass flasks (50 mL) in the MMG medium at a concentration of 10\u003csup\u003e6\u003c/sup\u003e conidia/mL, shaken at 37 \u0026deg;C during 24 h (\u003cem\u003eAfm\u003c/em\u003e) or 30 \u0026deg;C for 48 h (\u003cem\u003eNcr\u003c/em\u003e). Mycelia were collected as described above.\u003c/p\u003e\n\u003cp\u003eFor total lipid extraction, 50 OD\u003csub\u003e600\u003c/sub\u003ea pellet of PBS-washed \u003cem\u003eSce \u003c/em\u003ecells were resuspended in 0.5 mL of 120 mM Na-Acetate, pH 4.5. Then, 3.75 volumes of CHCl\u003csub\u003e3\u003c/sub\u003e:CH\u003csub\u003e3\u003c/sub\u003eOH (2:1) and 1 mL of glass beads (\u0026oslash; 0.25-0.5 mm, Roth) were added and cells disrupted through mechanical lysis using a FastPrep Instrument (MP\u0026trade; Biomedicals, Serial N\u0026deg; 10020698) at 1 min 5.5 m/s repeated 6 times with cooling on ice between each cycle. Cell lysates were incubated 3 h on a rotating wheel at 4\u0026deg;C. Then, 1.25 volumes of CHCl\u003csub\u003e3\u003c/sub\u003e and 1.25 volumes of 120 mM Na-Acetate pH 4.5 were added and the samples vortexed 1 min. Phases were separated by centrifugation (9000\u0026times;\u003cem\u003eg\u003c/em\u003e; 30 min; 4\u0026deg;C) and the lower organic phase containing lipids was transferred into a clean glass tube and dried under vacuum (SpeedVac vacuum concentrator). Drying was finalized under an argon flow. Lipids were stored at -20\u0026deg;C or resuspended in 50-100 \u0026micro;L of CHCl\u003csub\u003e3\u003c/sub\u003e:CH\u003csub\u003e3\u003c/sub\u003eOH (1:1, v:v) for analysis on TLC. In the case of \u003cem\u003eAfm \u003c/em\u003eand \u003cem\u003eNcr \u003c/em\u003estrains, mechanical cell disruption was performed as follows: 2g of fresh mycelia, dried on paper towel, were ground in a mortar with a pestle in the presence of liquid nitrogen and the resulting fine powder was resuspended in 1 mL (1 vol.) of 120 mM Na-Acetate pH 4.5 and treated as described above. For \u003cem\u003eAfm \u003c/em\u003econidia total lipids, 7-day old mycelial lawns grown on on malt agar plates were flooded with 10 mL of sterile H\u003csub\u003e2\u003c/sub\u003eO/0.05 % Tween-20 and conidia scrapped off the plate, filtered through a 40 \u0026micro;m cell strainer (ref. 0999225, Grosseron) and centrifuged 20 min, 8000x\u003cem\u003eg\u003c/em\u003e at 4 \u0026deg;C. The supernatants were discarded and spores treated as described for \u003cem\u003eSce\u003c/em\u003e cells.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e Lipid analysis by Thin-Layer Chromatography (TLC)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTLC plates (Silica gel 60 aluminum foils, Sigma-Aldrich, 10 x 10 cm) were used. Lipids in the CHCl\u003csub\u003e3\u003c/sub\u003e:CH\u003csub\u003e3\u003c/sub\u003eOH (1:1, v:v) solvent were spotted on TLC, typically, 10-20 \u0026micro;L of total lipids or 25 \u0026micro;L of radiolabeled lipids extracted from \u003cem\u003ein vitro \u003c/em\u003ereactions (see below). TLCs were developed with the CHCl\u003csub\u003e3\u003c/sub\u003e:CH\u003csub\u003e3\u003c/sub\u003eOH:H\u003csub\u003e2\u003c/sub\u003eO mobile phase (130:50:8) for 10 min, and air-dried. TLCs were stained with a sulfuric acid/MnCl\u003csub\u003e2\u003c/sub\u003e solution (concentrated sulfuric acid 9 mL, MnCl\u003csub\u003e2\u003c/sub\u003e.4H\u003csub\u003e2\u003c/sub\u003eO 0.8 g, CH\u003csub\u003e3\u003c/sub\u003eOH 120 mL, H\u003csub\u003e2\u003c/sub\u003eO 120 mL) or with ninhydrin (Sigma-Aldrich, 0.4 % w/v in EtOH 100 %, v/v) and heated at 100 \u0026deg;C, 15 min. Plates were imaged under white light or at 254 nm. Radiolabeled lipid species were revealed by exposing TLC plates onto a Fuji Imaging Plate and analyzed with a Typhoon TRIO, Variable mode imager (GE Healthcare).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eIn Vitro\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e lipid aminoacylation assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eReactions were performed in the following mix: 100 mM Na-HepespH 7.2 buffer containing 30 mM KCl, 12 mM MgCl\u003csub\u003e2\u003c/sub\u003e, 10 mM ATP, 0.1 mg/mL bovine serum albumin, pure yeast tRNA\u003csup\u003eAsp\u003c/sup\u003e (10 \u0026mu;M) (46), 10 \u0026micro;M [U-\u003csup\u003e14\u003c/sup\u003eC]-Asp (280 cpm/pmol, Perkin-Elmer, NEC268E050UC) in a final volume of 50 \u0026mu;L.To test the transfer of [\u003csup\u003e14\u003c/sup\u003eC]-Asp onto lipids, total lipids or pure sterols were added to a finalconcentration of 2 mg/mL, and commercial pure sterols were added to a final concentration of 0.5 mg/mL.Purified ErdSDN86 was added to initiate the reaction before a 45-min-long incubation at 30 \u0026deg;C. After incubation, reactions were stopped with addition of 500 \u0026mu;L ofCHCl3:CH\u003csub\u003e3\u003c/sub\u003eOH:120 mM Na-acetate pH 4.5 (130:50:8, v/v/v) and vortexing. Then, 130 \u0026mu;L of CHCl\u003csub\u003e3\u003c/sub\u003e and 130 \u0026mu;L of Na-acetate 120 mM pH 4.5 were successively added, and the mixture was vortexed and centrifuged for 1 min at 5,000 \u0026times; g (RT). The lower organic phase was recovered and dried under vacuum. Reaction products were then dissolved in a CHCl\u003csub\u003e3\u003c/sub\u003e:CH\u003csub\u003e3\u003c/sub\u003eOH (1:1, v:v) mixture, spotted on TLC plates and separated with the CHCl\u003csub\u003e3\u003c/sub\u003e:CH\u003csub\u003e3\u003c/sub\u003eOH:H\u003csub\u003e2\u003c/sub\u003eO mobile phase. TLC plates were exposed onto an imaging plate (Fuji Imaging plate) for at least 2 h. Radioactivity was detected using a Typhoon TRIO variable Mode Imager (GE Healthcare). Quantification of radioactivespots or Erg-Asp and phosphoethanolamine (PE) bands was performed using the ImageJ software (number of pixels).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e Protein extraction and Western blots\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eYeast total proteins from \u003cem\u003eSce\u003c/em\u003e were extracted using 1 OD600nm of cells that were resuspended and incubated 10 min in 500 \u0026mu;L of pre-cooled NaOH 0.185 N, then precipitated by adding 50 \u0026mu;L of Trichloroacetic acid (TCA) 100 % and incubated 10 min on ice. Finally, the samples were centrifuged at 13, 000 x \u003cem\u003eg \u003c/em\u003efor 15 min and the resulting pellets were resuspended in 100 \u0026mu;L of Laemmli Sample Buffer. Then, 8 \u0026mu;L of each sample was resolved on 10 % SDS-PAGE gels. Samples were separated by using a BioRad Mini-PROTEAN electrophoresis apparatus.For western blotting, proteins were transferred onto PVDF membranes that were blocked in 5% (w/v) skimmed milk in TBS-Tween (TBS 1X, Tween-20 0.3 % (v/v)) for 1 h at RT. Primaryantibodies (polyclonal anti-DUF2156, Covalab, France, anti-PGK, dilution 1/10,000) were incubated overnight at4 were incubated overnight a times with TBS-Tween. Membranes were then incubated for 1 hwith HRP-conjugated secondary antibodies (Goat anti-rabbit for anti-DUF2156 and Goat antimousefor anti-PGK) at RT. Revelation was performed with the BioRad clarity western ECLKit and monitored in a BioRadChemiDocTouch\u0026reg;apparatus.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e ErdS sequence alignments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe sequence of \u003cem\u003eAfm\u003c/em\u003eErdS was used as the probe to search homologs using BLAST with the default parameters. Then, 98 homologous sequences were selected across Dikarya and aligned using MUSCLE\u003csup\u003e30\u003c/sup\u003e, and the resulting alignment was used to produce a sequence logo at WebLogo 3 (https://weblogo.threeplusone.com/create.cgi).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe atomic models have been deposited in the PDB under the accession codes #### (ErdS tetramer), #### (ErdS/tRNA dimer on tRNA located AspRS) and #### (ErdS/tRNA dimer on tRNA located ATT). The cryo-EM density map has been deposited in the Electron Microscopy Data Bank under the accession codes EMD-##### (ErdS tetramer), EMD-##### (ErdS/tRNA dimer on tRNA located AspRS) and EMD-##### (ErdS/tRNA dimer on tRNA located ATT). 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[20] In vitro mutagenesis and plasmid shuffling: From cloned gene to mutant yeast. in 302\u0026ndash;318 (1991). doi:10.1016/0076-6879(91)94023-6.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-7291846/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7291846/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eErgosteryl-3β-O-L-aspartate synthase (ErdS) catalyzes tRNA-dependent aspartylation of ergosterol, a lipid essential for fungal cell membrane integrity. However, the functional significance of ergosteryl-aspartate and the molecular mechanisms underlying its synthesis remain unclear. Here, we show that ErdS localization is highly dynamic and provide evidence that Erg-Asp is required for proper hyphal growth, sporulation, and spore germination, and likely influences stress tolerance. The cryo-electron microscopy structure of ErdS revealed an unprecedented sterol-binding pocket. In addition, the structures in complex with non-hydrolyzable Asp-N-tRNA\u003csup\u003eAsp\u003c/sup\u003e uncovered a tRNA-guided intramolecular aminoacyl transfer mechanism between two functional domains of the enzyme. The CCA end of tRNA\u003csup\u003eAsp\u003c/sup\u003e undergoes a large displacement to reach the aa-tRNA transfer active site, while the tRNA elbow is clamped by a long extension of the N-terminal α-helix. The present structural and mutational analyses demonstrate that domain fusion, dynamic repositioning, and tRNA-mediated substrate handover underlie the multifunctional catalytic efficiency of ErdS and facilitate Erg-Asp synthesis independently from protein synthesis. These findings elucidate the unique regulatory mechanism of tRNA-dependent sterol modification and provide insights into fungal membrane dynamics, highlighting potential novel targets for antifungal therapies.\u003c/p\u003e","manuscriptTitle":"Structural basis for tRNA-dependent sterol aminoacylation underlying cell membrane integrity","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-22 05:31:39","doi":"10.21203/rs.3.rs-7291846/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"5bedf02b-3edb-4238-a31d-48d00c4d5d42","owner":[],"postedDate":"August 22nd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":53465807,"name":"Biological sciences/Structural biology/Electron microscopy/Cryoelectron microscopy"},{"id":53465808,"name":"Biological sciences/Cell biology/Mechanisms of disease"}],"tags":[],"updatedAt":"2026-04-24T13:45:43+00:00","versionOfRecord":[],"versionCreatedAt":"2025-08-22 05:31:39","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7291846","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7291846","identity":"rs-7291846","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}