Sequential conformational transition of ArnB, an archaeal ortholog with Sec23/Sec24 core motif

Sequential conformational transition of ArnB, an archaeal ortholog with Sec23/Sec24 core motif | 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 Sequential conformational transition of ArnB, an archaeal ortholog with Sec23/Sec24 core motif Lars-Oliver Essen, Lukas Korf, Wieland Steinchen, Mohamed Watad, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3955852/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 ArnA and ArnB serve as regulators within the archaellum regulatory network by affecting the levels of archaellum components ArlB and ArlX in response to nutrient cues. Together, they form either a loose or a tight complex, whose transition is directed by phosphorylation via the kinase ArnC. For a structure-based analysis of this transition we solved a cocrystal structure of the ArnA/ArnB complex revealing that the zinc finger domain of ArnA interacts with the β-sandwich and C-terminal domain of ArnB. HDX data corroborate the phosphorylation-dependent transition from loose to tight ArnAB complexes. This transition depends on a structural transformation of ArnB by sequential phosphorylation, exposing the interaction surface of the C-terminal domain of ArnB for the forkhead-associated domain of ArnA. Furthermore, we found a striking structural similarity between ArnB and the membrane-curving proteins of the COPII vesicle system, Sec23/Sec24. The common Sec23/Sec24 core motif can be found in all domains of life, where it can apparently adopt a multitude of different functions. Overall, this implies that Sec23/Sec24 orthologs with a function in vesicle formation arose in Lokiarchaeota from related, but not necessarily functionally linked relatives as found in TACK Archaea. ArnB Sec23/24 sequential phosphorylation order to disorder bidirectional transition Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction The archaellum serves as the primary motility apparatus in Archaea, facilitating self-locomotion and enabling adaptive habitat transitions 1 . Its responsiveness to diverse environmental stimuli is governed by a regulatory network known as the archaellum regulatory network (Arn) 2 . Within this intricate system, which regulates transcriptional and translational adaptations, the proteins ArnA and ArnB play pivotal roles in the Crenarchaeon Sulfolobus acidocaldarius , particularly in the regulation of arlB (formerly flaB ), the gene encoding the archaellin 3 – 5 . In the context of nutrient availability in S. acidocaldarius , ArnA and ArnB orchestrate the regulation of arlB 6 . Notably, the von Willebrand factor domain-containing ArnB forms a complex with the forkhead-associated domain (FHA) protein ArnA 5 . Deprivation of either ArnA or ArnB results in an upregulation of arlB expression, leading to hypermotility in the organism 6 . Furthermore, the interplay between nutrient levels and the formation of the ArnAB complex affecting the formation of archaella has been elucidated 5 . Starvation conditions induce a loss of interaction within the complex, subsequently promoting the expression of arlB . Consequently, ArnA and ArnB are identified as negative regulators of the archaellum when complex formation is established. Phosphorylation-dependent interactions between ArnA and ArnB are facilitated by the phosphatase PP2A and the kinase ArnC, maintaining a delicate balance between complex formation (phosphorylation) and dissociation (dephosphorylation) 7 . Despite in-depth in vitro and in vivo studies, the exact interaction mode of ArnA and ArnB, purportedly located in the C-terminal region of ArnB, remains elusive. Moreover, the mechanism by which the ArnAB complex controls the ArlB levels remains enigmatic. The formation of ArnAB oligomers that depends on phosphorylation is another feature 7 . Intriguingly, we found that the structural characteristics of ArnB with its intriguingly associated β-sandwich, von Willebrand factor type A (vWFA) and α-helical domains form a structural blueprint widely distributed among all domains of life, which also includes the eukaryotic COPII-vesicle assembly factors Sec23/Sec24. However, functional divergence as observed in archaeal proteins like the histones 8 , which are functionally distinct in eukaryotes despite sharing a common core fold, illustrates the limitation of relying solely on structure-based function inference. Given this caveat we performed a biochemical and structural analysis of ArnAB interaction that revealed a novel mode of sequential conformational change of its Sec23/Sec24 core motif by phosphorylation. Results Overall structure of the ArnA-ArnB complex and structural comparison with Sa vWA2 We were able to solve the crystal structure of the vWA2 paralog 5 and archaellum regulating factor ArnB (UniProt: Q4J9H3, saci_1211) in its complex with ArnA (UniProt: Q4J9H4, saci_1210). The ArnAB cocrystals comprise two complexes per asymmetric symmetry unit, whose structures were solved by molecular replacement and refined at 2.5 Å resolution. The ArnAB complexes are defined by electron density for residues T2-S380 of ArnB and the zinc-finger (ZnF) domain of ArnA (P16-K42, Fig. 1 A, PDB: 8S05). The latter implies a loss of the FHA domain and the linker region of ArnA, possibly by unspecific proteolysis as observed before when solving the structure of the ArnA FHA domain 5 . The overall architecture of ArnB corresponds mostly to its paralog, the van Willebrandt Factor A (vWFA)-containing protein Sa vWA2, including the vWFA domain, an eight-stranded β-sandwich whose topology is split by the vWFA domain, as well as the C-terminal, four helix bundle motif, that has been classified as ArnB_C domain (InterPro entry IPR040929) (Fig. 1 A) 2 , 5 . In contrast to Sa vWA2, the ArnB_C domain of ArnB harbors an elongated helix pair at the terminal region of the motif, revealing one of the most substantial structural differences between ArnB and Sa vWA2 (Fig. 1 D). This extension presents additional threonine residues that allow for potential interaction of the forkhead-associated (FHA) domain of ArnA, known for its ability to bind phospho-threonines, with ArnB (Fig. 1 D right panel) 9 . Moreover, ArnB features an additional helix (P95-Q103) in the vWFA domain between β5 and α2 of Sa vWA2 increasing the total helix count to ten (Fig. 1 D). The vWFA domain of ArnB also harbors a Na + ion coordinated by D46, S50, T110, T135 and D136 in the metal ion-dependent adhesion site (MIDAS, Fig. 1 C). While ArnA interacts strongly with its FHA domain to phosphorylated ArnB (see below), the zinc finger domain (ZnF domain) itself is sufficient to promote an interaction without a post-translational modification of ArnB. This ZnF domain belongs to the RanBP2-type (IPR001876) and is characterized as a ZnF ribbon domains by two consecutive, distorted β-hairpin motifs, which together a zinc ion via C21, C24, C35 and C38. The interface of the ZnF domain of ArnA with ArnB has a rather moderate size of 504/574 Å 2 for ArnAB chains A/C and B/D, respectively. The interactions are mostly of hydrophobic nature and include the C-terminal β-hairpin motif (D31-Q41) of the ZnF domain as well as the N-terminal β1-β2 loop (H12-K21) of the β-sandwich domain, it’s α-helical linker to the ArnB_C domain (V286-I293) and adjacent residues of the ArnB_C domain facing the ZnF domain. Accordingly, the ArnA-ArnB interaction based on the ZnF domain appears to be rather weak as indicated by pulldown assays (Figure S1 A), but traceable by mass photometry 7 . A further point for the uniqueness of the ZnF-mediated ArnA-ArnB interactions are AF2-multimodels, which were unbiased of the ArnAB structure and show almost an identical interaction as in the ArnA-ArnB cocrystal structure for 4 of the 5 predicted ArnA-ArnB models (Figure S2) with displacement r.m.s.d. values of 1.57–1.65 Å for M1-Q25 of the ZnF domain. This indicates that the intrinsic sequence covariation for ArnA and ArnB domains is already significant enough to provide a robust indicator for the relatively small ArnB/ZnF domain interface. Moreover, an ArnA-ArnB interaction is also displayed in solution by SAXS data (Fig. 1 E) as the ab initio envelope as derived from the SAXS data is fittable to the ArnA-FHA and ArnAB crystal structures. Here, the pair distance distribution function P(r) suggests an overall elongated shape (Figure S3) and thereby supports an ArnA-ArnB interaction based on the ZnF domain, as in the crystal structure, with a flexible region followed by an unbound FHA domain due to a lack of pThr anchor points. Promiscuity of ArnB phosphorylation-dependent interaction sites In the search for the entire interaction site of the strong ArnA-ArnB interaction based on phospho-threonine interaction, we performed a comprehensive mass spectrometry-based analysis. Notably, there are many potential interaction sites found in ArnB, especially in the C-terminal HTH motif (Fig. 2 A) where interaction appears to be most likely, based on structural analysis. Accordingly, we first opted to investigate the phosphorylation pattern by the kinase ArnC (UniProt: Q4J9J0, Saci_1193) with the previously reported phosphorylation conditions 7 . Interestingly, we found that many threonines in the ArnB_C domain were phosphorylated during our in vitro phosphorylation, raising some interesting questions about the native phosphorylation conditions. However, as 60 minutes of incubation time at 55°C yielded many different potential interaction sites we opted to find the ones that are phosphorylated first. Hence, we investigated the effect of different incubation times on the phosphorylation pattern of ArnB. In time points of 5 min, 15 min, 30 min and 60 min (resembling our reference sample) the phosphorylation pattern of ArnB was analyzed in a tryptic digest MS experiment. Interestingly, ArnA could still be efficiently pulled down along with ArnB during purification with phosphorylation time of little as 5 min. Moreover, phosphorylation of threonines located closer to the N-terminal side of the ArnB_C domain takes place only after T353, T354, T359, T363 and T375 are phosphorylated and only until T322, which aligns with the known phenomenon that multiple phosphorylation usually occur as clusters in a protein 10 . Accordingly, these main threonines are phosphorylated after 5 minutes already, while other phosphorylated threonines that have potential counterparts in vWA2 are found phosphorylated only after 15 minutes or more incubation time. This may indicate ordered processivity of hyperphosphorylation, like in the cyclin–Cdk1–Cks1 system 11 and goes along with the threonines being located in the extension of the C-terminal HTH motif of ArnB, besides T375, which are therefore not present in vWA2. Additionally, it is reported that vWA2 does not interact with ArnA 6 , supporting the assumption that the C-terminal HTH extension is the main interaction side for the ArnA FHA domain. As this narrowed down the possible interaction sites, we attempted to identify the exact position of the ArnB-FHA interaction by a comprehensive alanine mutagenesis study. Inducing the mutation of either T353A + T354A or T359A + T363A it was not possible for the FHA domain to interact with ArnB anymore, if phosphorylated for 5 min or less. However, increasing the phosphorylation time to 60 min, ArnA could be pulled down with ArnB without issues again. Additionally, even when threonines that were found to be phosphorylated only after 5 min were mutated as well, ArnA could still be pulled down along with ArnB, represented by the ArnB-T343A-T344A-T353A-T354A-T359A-T363A-T371A-T375A mutant (Figure S1 B). In general, this gives an idea of the more likely interaction side of the FHA domains location at the C-terminal elongation of the ArnB_C domain, but leaves it open if multiple FHA interaction sites play a role in the interaction of ArnA and ArnB. However, the hyper-phosphorylation we observed leads to multiple structural rearrangements, as discussed in the following, and hence are not necessarily interaction sites but structural factors important for a correct ArnB-FHA interaction. HDX-MS reveals phosphorylation-dependent structural relieves of ArnB In order to get insights into the interaction site(s) between ArnA and phosphorylated ArnB in solution, we made use of the property of amide protons to exchange for protons from the aqueous solvent, the extent of which was traced upon incubation of the proteins in deuterated buffer. This hydrogen/deuterium exchange (HDX) was then, after digestion of the proteins into peptides, quantified by mass spectrometry (MS). We subjected ArnA, ArnB, phosphorylated ArnB, and the ArnA/phosphorylated ArnB complex to HDX-MS experiments, allowing us to resolve, i) the conformational changes that ArnB undergoes upon phosphorylation, and ii) the regions of ArnA and phosphorylated ArnB establishing the interaction interface in their complex. For ArnA and ArnB, we could identify 80 and 145 peptides, respectively, that covered more than 90% of their corresponding amino acid sequences (Figures. S1A and S2A, Supplementary Dataset 1). The HDX profile of ArnA corroborated its predicted domain topology, in particular the disordered nature of the linker (S28-N99, where maximal HDX was reached after 10 s of deuteration) joining the N-terminal ZnF domain to the C-terminal FHA domain (Figure S4B). Upon complex formation of ArnA with phosphorylated ArnB, HDX reduction became apparent in both the ZnF domain and FHA domains thus marking the major sites of interaction for ArnA (S1C-D). Specifically, the phosphate recognition module of the FHA domain constituted by R132 and R147 exhibited the strongest HDX reduction consistent with their role in FHA-mediated phosphate recognition for strengthening the ArnA/ArnB interaction 5 . The HDX profile of ArnB itself primarily showed regions of higher-order structure (low HDX at 10 s of deuteration and progression in HDX over the time-course) and only small disordered areas coinciding with short linkers of the crystal structure (Figures S2B, Fig. 1 A). However, phosphorylation of ArnB induces widespread conformational changes, according to HDX increases of the ArnB domain, in parts of the β-sandwich and vWFA domains vicinity (Fig. 3 B). These changes may reflect either a partial unfolding event or a disentanglement of the three associated domains. Furthermore, binding of ArnA to phosphorylated ArnB, in turn, reduced the observed HDX rates in proximity of the ArnA-ZnF domain binding site (Fig. 3 A). Notably, ArnB residues D295-A310, constituting the N-terminal portion of helix α7, incorporate more deuterium upon phosphorylation, whereas a reduction was apparent upon ArnA binding (Figure S6). It may be hence speculated that the phosphorylation-induced conformational change could be a prerequisite for tight ArnA binding. Likely due to the phosphorylation of the threonine side chains, no peptides covering the C-terminus’ residues T353 onwards could be retrieved thus precluding further conclusions on this presumed ArnA-FHA domain interaction site by HDX-MS of ArnB (Figure S5A). Overall, HDX-MS corroborates the binding site of the ArnA-ZnF domain on ArnB observed in the complex structure (Fig. 1 ) and provides evidence for a secondary interaction site established with the ArnA-FHA phosphate recognition module. tims-ToF proteomics data for ArnA and ArnB deletion strains Although the roles of ArnA and ArnB in the archaellum regulatory network are established 5 , 6 , the mechanism by which regulation occurs under nutrient limitation remains undisclosed. Consequently, we analyzed the Sulfolobus acidocaldarius proteome of the ΔarnA and ΔarnB strains in two nutritional states in comparison with the respective wild type (WT). For this analysis biological triplicates were grown of each strain, and samples were taken under nutrient rich and starved conditions. Samples were measured in a timsTOF (trapped ion mobility spectrometer) mass spectrometer and quantified via label free quantification before averaging the technical duplicates of each sample and further analysis. These measurements led to the identification of 1,699-1,710 proteins per sample leading to the identification of 1,723 overall proteins of the 2,222 gene products known for the respective Sulfolobus acidocaldarius strain (Supplementary Dataset 2). The comparison of the knockout strains with their respective WT sample reveals 1,713-1,716 identified proteins per comparison with overlapping count of 1,694-1,704 (98.7–99.4%). As the overall proteome is basically unaffected by the deletions, we investigated the effects of the deletion strains under higher stringency i.e. a two-tailed t -test with a p-value cutoff of < 0.05. This analysis revealed 325(rich)/384(starved) statistically changed proteins compared to the WT proteome for the ArnA knockout and 398(rich)/510(starved) for the ArnB knockout, respectively. In addition, we also included the stringency factor of a fold change of at least 50% to not only check for statistical relevance but also for biological effects (Fig. 4 A/B). As displayed in the volcano plot analysis (Fig. 4 A/B, S8-9), this revealed 115(rich) / 243(starved) proteins for the ΔarnA and 185(rich) / 279(starved) proteins for the ΔarnB strain passing that high stringency test. This shows that under starvation conditions the effect on the proteome level is more prominent under nutrient starved conditions for both knockouts. Moreover, the effect of the ArnB knockout is also slightly more impactful on overall altered proteins both after t-testing and after employing the additional fold change cutoff of 50%. To see if these statistically relevant changes on the proteome level also reflect on the biology of S. acidocaldarius , we conducted an intensive gene ontology (GO) term analysis (Fig. 4 C/D). Interestingly, this analysis showed that both knockouts have a preference for metabolic enzymes, especially for additional nitrogen containing amino acids pathways. Nevertheless, purine/nucleotide, acetyl-CoA and carbohydrate metabolism enzymes were also significantly enriched after knockout of either ArnA or ArnB. Despite a small difference in statistical significance and actual enrichment score the GO term analysis delivered quite similar results for both knockouts. Notably, when comparing both knockouts with each other, only 43–61% of the significantly altered proteins can be found in both deletion strains simultaneously (Fig. 4 G). However, those significantly altered proteins that can be found in both knockouts do share a very high correlation of 82–86% (Fig. 4 E/F). Together this leads to the conclusion, that around half of the impact of knocking out either ArnA or ArnB is apparently based on the interaction of them with each other to some degree. Vice versa this means that the other half of the effected protein levels are apparently independent from the ArnA-ArnB interaction or at least without direct correlation. A comparison of these proteomics data with a deletion variant of S. acidocaldarius that missed the GPN-loop GTPase Sa GPN and exhibited diminished motility 12 shows that the proteome changes of the hypermotile ArnA and/or ArnB deletion variants 13 are more modest. This suggests a more intimate involvement of ArnA and ArnB in the regulation of motility than of Sa GPN, which causes large-scale changes in the proteome network. Accordingly, there is no correlation for significantly altered protein levels between the Sa GPN knockout and the ArnA or ArnB knockouts (Figure S10). This suggests that the role of ArnA and ArnB in the archaeal regulatory network is independent of Sa GPN. Evolutionary context of ArnB, which contains the Sec23/Sec24-core motif In the current state of literature, the function of ArnB, a homolog of the vWA2 protein in S. acidocaldarius 5 , has been associated with the archaellum regulatory network. It has been reported that ArnB is a negative regulator of the archaellin arlB, regulated by the interaction with its partner ArnA. However, during investigation of ArnB we found structural similarities with the membrane curvature proteins Sec23/24, which are central components of the assembly machinery for eukaryotic COPII vesicles. Sec23/24 harbor a domain arrangement highly reminiscent of ArnB and even include an N-terminal ZnF domain like ArnA. Accordingly, Sec23/24 contain as a core motif the vWFA, β-sandwich and C-terminal helical domain, the latter being similar to the ArnB_C domain, besides an additional C-terminal domain (Fig. 5 A). Superposition of the Sec24 core motif (PDB: 1m2v, P301-I749 of chain B) and ArnB reveals a structural deviation of 5.7 Å for 302 Cα atoms. Here, differences between the Sec24 core motif and ArnB are mostly found for the length of helices in the helix pair α9/α10 of the ArnB_C domain and the relative orientation of the β-sandwich. Notably, the ZnF domain of Sec23/Sec24 and that of ArnA in the ArnAB complex pack to different sites of the β-sandwich of the core motif. In the ArnAB complex structure, the ZnF domain occupies a site made up by an edge between the β-sandwich and the ArnB_C domains, whereas in Sec23/Sec24 the ZnF-domain associates to the opposite site of the β-sandwich domain (Fig. 5 B). Another feature in this context is the packing between the Sec23/Sec24 core motif and the C-terminal domain. Although ArnB lacks the gelsolin-type C-terminal domain of Sec23/24, the FHA domain of ArnA is predicted to interact in a phosphorylation-dependent manner with the α-helix domain that resembles the packing of the Sec23/Sec24 motif and the C-terminal domain. The close relationship between the ArnAB assembly and the eukaryotic Sec23/24 core motif prompt for a wider distribution of the Sec23/Sec24 core motif within the domains of life. Using foldseek and the ArnB crystal structure we found a wider occurrence for the ArnB/Sec23 core motif (Supplementary Dataset 3) than previously suggested by the structural relationship between vWA2 from S. acidocaldarius and a vWA protein from the actinobacterium Catenulispora acidiphila 5 . Structural orthologs are found in other bacterial phyla than the actinobacteria. For example, Escherichia coli has a predicted structural ortholog, YfbK, whose domain topology is found in ~ 5900 gene products (status 01/24, Figure S11A). Besides matching Alphafold2 models from Archaea including Euryarchaeota like Halorubrum we also find Sec23/Sec24 core motifs in other Korarchaeota and Heimdallarchaeota (Figure S11B). More surprisingly, the closest structural hits outside the archaeal domain are found in the plant and fungal kingdoms despite marginal sequence identities. Orthologs in plants like Arabidopsis thaliana and Zea mays (Figure S11C) currently lack an assigned function. Notably, foldseek hits in the animal kingdom, apart from the expected Sec23/Sec24 orthologs, lack a direct structural relationship to the Sec23/Sec24 core motif and ArnB (Figure S11D). Discussion This work presents much needed structural information, supported in both solution and crystal structure, of the ArnAB complex and features more information about its in vivo role. The role of ArnA, ArnB and its hetero complex has so far been associated with the archaellum regulatory network, hence the name Arn 5 , 7 . Nevertheless, their biological role is not yet fully understood leaving open questions of why they are found in exosomes and why they share a structural homology with the Sec23/24 complex. Recently, it was reported that the ArnA homolog from Saccharolobus islandicus ( Sis ) interacts with promotor sequences supporting a transcription factor role for ArnA 14 , although DNA binding was rather unspecific. However, during our work, we could not find any support that ArnA from S. acidocaldarius ( Sa ArnA) itself is capable of binding DNA. Moreover, there are no structural indications that either ArnB or ArnA have DNA binding sites, especially since the ZnF domain of ArnA, which would be an obvious target, is part of a protein-protein interface. As Sis ArnA was purified from the native organism, its DNA binding activity may depend on additional interaction partners than ArnB or post-translational modifications, which require further elucidation. Furthermore, our comprehensive proteomic data suggests involvement of ArnA and ArnB in anabolic metabolism pathways. Accordingly, knockout of either of them lead to a gene enrichment in enzymes concerning amino acid metabolism, especially N-rich amino acids, carbohydrates and acetyl-CoA metabolism. This is particularly resembled by changes in protein levels of the Gln-synthase (Q9HH09), succincyl-CoA ligase (Q4J9C0) or an MFS sugar transporter protein (Q4J7I0). Obviously, the known overproduction of the archaellum, an indicator of hypermotility, caused by the ArnA/ArnB knockouts 6 , indicates per se changes of the metabolic repertoire in S. acidocaldarius , when switching from a an immobile, sessile form to a planktonic lifestyle. Compared to other proteomic data regarding proteins that are involved in the Arn-system like the GPN-loop GTPase from S. acidocaldarius Sa GPN, these changes are completely different 12 . While only half of the statistically relevant proteome changes is common between ArnA and ArnB deletion strains, the intrinsic connection between both is also mirrored by the nearly symmetrical alterations observed in proteins that play pivotal roles in both strains. Therefore, we are confident to say that the metabolic impacts we observed are directly related to the specific knockouts. Accordingly, the large-scale proteome impacts observed for the Sa GPN knockout appear to be particularly more random. Notably, as Sa GPN is being considered part of the Arn system, physical association of the ArnAB complex with Sa GPN might link to a collaborative function of all three of them. This could also explain why the previously reported PP2A co-IP assay 7 pulls down not only ArnA and ArnB but Sa GPN as well. However, it may also be of coincidental nature and no physical association connects the ArnAB complex with Sa GPN. Nevertheless, the aspect of the ArnA-ArnB interaction corresponds to a ‘flip switch’ depending on the phosphorylation state of ArnB and its respective oligomeric state. These states are regulated by the PP2A and ArnC phosphatase-kinase system and induce heterodimerization as well as higher oligomerization upon threonine phosphorylation based on the FHA-pThr interaction. Interestingly, the oligomerization processes possibly involve a (partially) unwinding or loosening of the ArnB motifs. Accordingly, the stand-alone domain of PP2A exhibits a more restricted array of control modes compared to ArnC, which exhibits an elongated N-terminal region containing a TPR-like helical domain in addition to its C-terminal Ser/Thr kinase domain and thereby offering a more diverse range of regulatory mechanisms. The Sec23/24-like domain architecture of ArnB with its β-sandwich, vWFA and C-terminal helical bundle domain is conserved among all domains of life. Captivatingly, the evolutionary connections between different kinds of proteins within all three domains of life is apparent in their structure with only moderate changes. However, due to a weak phylogenetic connection between them the functions of the eukaryotic Sec23/24 exert completely different functions than their archaeal predecessors. Therefore, the structural homology between ArnB features with Sec23/24 does not necessarily place ArnB with the COPII vesicle system, as structural homologs of ArnB can even be found in Escherichia coli (Figure S11A). Nevertheless, two main scenarios appear likely: function in vesicle transport either arose late, i.e. in Asgard Archaea with their symbiotic lifestyles with bacteria, or upon the transition to Eukaryotes, i.e. during the manifestation of endosymbiosis. Moreover, a prerequisite for a functional switch may be the association with exosomes as observed before 15 , even when ArnA/ArnB are not directly involved in exosome formation. Interestingly, ArnB demonstrates a phosphorylation-dependent alteration of its conformational flexibility as revealed by HDX-MS. Phosphorylation proceeds at multiple, but specific sites and follows a pattern consistent with a model in which ArnC-mediated phosphorylation starts at threonines located in the tip regions of the C-terminal helical bundle (T353-T363). Subsequently, it propagates to threonine sites, which are mostly buried within the helix bundle/vWFA domain interface (T371-T375), and finally extends to sites closer to the N-terminus of the helix bundle domain. It is worth noting that such a sequential phosphorylation and associated partial unfolding of the protein is not without precedence 16 , 17 . For example, for p19 INK 4 d , an ankyrin-repeat protein, it is reported that the sequential phosphorylation within the helical repeat domain progressively destabilizes the N-terminal half of the protein, ultimately leading to the unfolding of the corresponding section 16 . Moreover, the casein kinase 1a (CK1a) of Neurospora progressively hyper-phosphorylates the clock protein FREQUENCY (FRQ), which triggers a conformational change driven by clustered phosphorylation 17 . In general, multi-phosphorylation exerts regulatory influence over protein structures, facilitating bidirectional transitions between ordered and disordered states 18 . Notably, the sequential part of the phosphorylation patterns apparently does not necessarily affect ArnB-FHA interaction, as different threonine mutants of ArnB were still able to bind the FHA domain of ArnA, as long as alternative phospho-sites were available (Fig. 2 C/D). However, these large-scale conformational changes observed in ArnB align with a dual-point attachment of ArnA facilitated by both its ZnF and FHA domains. This interaction could serve as the catalyst for the assembly of larger molecular complexes involving ArnA and ArnB, resembling but not identical to the established Sec23/Sec24 COPII coat formation. Many of the intricacies surrounding the functionality and assembly of the ArnAB complex stem from uncertainties related to the oligomerization behavior, as well as the precise interaction point of the ArnA FHA domain and the role of the ArnA linker domain. The absence of structural support has led to various plausible scenarios. Consequently, we posit three primary operational modes governing the heterocomplex formation of ArnA and ArnB, based on our structurally supported findings. Firstly, these scenarios encompass the possibility of simple heterodimerization, where observed oligomers may lack biological relevance. This could be attributed to their potentially artificial nature in vitro , stemming from a deficiency in regulated phosphorylation, underscored by the varying incubation times with ArnC, as illustrated in this and previous studies 7 (Fig. 6 top right). Secondly, it's conceivable that another element plays a role in ArnAB function. Both DNA and other proteins, such as Sa GPN and Sa UspA, identified in the previously reported co-IP assay on PP2A interaction partners, emerge as potential targets (Fig. 6 mid right). Lastly, polymerization based on the FHA domain appears probable. Numerous phosphorylated states of the ArnAB complex 7 , especially with extended ArnC incubation times, hint at a multifaceted interplay facilitated by the FHA domain-pThr interaction, thereby mimicking a Sec23/24-like behavior (Fig. 6 bottom right). In conclusion, the intriguing phosphorylation-dependent interaction between the archaellum regulatory network proteins, ArnA and ArnB, not only adds to our understanding of archaellum-related functions but also positions them as potential precursors to COPII homologs. This suggests a broader significance, possibly making them among the earliest COPII-like proteins, which were already present in TACK Archaea. Methods Materials If not stated otherwise chemicals and materials were obtained from Carl Roth. Primers were ordered from Microsynth. Protein expression Overexpression of recombinant proteins of this study was performed in LB medium (10 g/L tryptone, 10 g/L NaCl, 5 g/L yeast extract) supplemented with 34 µg/mL chloramphenicol, using BL21(DE3) Rosetta cells. After induction of gene expression (0.5 mM IPTG at OD 600nm = 0.6–0.8) cultures were incubated overnight (16°C, 150 rpm, 18 h). Harvesting (20°C, 5,000 rpm, 20 min) was followed by resuspension in lysis buffer (150 mM NaCl, 50 mM Tris, pH = 8.0). Cell pellets were either used freshly for protein purification or frozen in liquid N 2 and stored at -80°C for later use. Protein purification without phosphorylation Cell pellets were lysed using a micro-fluidizer (2 min/2L cell pellet equivalent) after harvesting. The lysate was centrifuged (18,000 rpm, 20°C, 20 min), the supernatant heat-treated (70°C, 20 min) and centrifuged again (18,000 rpm, 20°C, 20 min) before being loaded to a Ni-NTA column (5 mL, Cytiva). After sample application, the column was washed with 9 column volumes wash buffer (150 mM NaCl, 50 mM Tris, 25 mM imidazole, pH = 8.0) and eluted with 5 column volumes elution buffer (150 mM NaCl, 50 mM Tris, 500 mM imidazole, pH = 8.0). The elution was concentrated (< 2.5 mL) and applied to a HiLoad® 16/60 Superdex® 200 pg (Cytiva) size-exclusion column, which had been equilibrated with running buffer (150 mM NaCl, 50 mM Tris, pH = 8.0). Fractions containing the proteins, validated by SDS-PAGE, were collected, concentrated, frozen in liquid N 2 and stored at -80°C. Purification of the ArnAB complex - Phosphorylation of ArnB For the generation of the phosphorylated ArnAB complex a 1L cell pellet of untagged ArnC, which was expressed and harvested as described above, was added to the ArnA-ArnB coexpression cell pellets before lysis. In addition, after the heat step ATP (Sigma Aldrich) and MnCl 2 (2.5 mM final concentration each) were added to the supernatant before incubation in a water bath (55°C, 5–60 min). Purification of the phosphorylated complex was performed with Ni-NTA affinity chromatography and SEC as described above. Crystallization and structure determination of the ArnAB complex Freshly purified, equimolar protein of ArnA and ArnB (4.85 mg/mL) was filtered with a centrifuge reaction tube micro filter (Merck) prior to crystallization experiments. Initial crystal hits of the ArnAB complex grew in well F11 of the JCSG Core III Suite (NeXtal) but were unsuitable for structure determination. Accordingly, a fine screen hanging drop vapor diffusion experiment on 24-well plates by mixing 1 µL of crystallization condition with 1 µL protein solution was conducted. The final monoclinic crystals grew in the presence of 0.17 mol/L ammonium acetate, 0.085 mol/L sodium citrate pH 6.0, 25.5% (w/v) PEG 4000, 16.5% (v/v) glycerin after 5 months. ArnAB data set was collected at the Swiss Light Source at Paul Scherrer Institute ( Switzerland ). Structure determination of the ArnAB complex was done by molecular replacement with the vWA2 structure (PDB: 5A8J) from Sulfolobus acidocaldarius as search model. Afterwards, the structure was refined by multiple rounds of manual structure building with Coot 19 followed by phenix.refine 20 , which also led to electron density defining the ArnA ZnF-domain. Data collection and refinement statistics can be found in Table S2. Protein structure was deposited to the Protein Data Base and can be accessed via the code: 8SO5. Small angle X-ray scattering (SAX) SAXS datasets were collected at the synchrotron Bio-SAXS beamline BM29 (ESRF) Grenoble, France 21 . The wavelength λ = 1.0 Å and the sample-to-detector distance of 2.43 m resulted in scattering vectors, q, ranging from 0.0025 Å −1 to 0.50 Å −1 . The scattering vector is defined as q = 4π sin θ / λ , where 2θ is the scattering angle. All experiments were performed at 20°C and the data were initially processed by the ATSAS software package 22 . SAXS data collection was performed in 150 mM NaCl, 50 mM Tris (pH 8) buffer. One-dimensional datasets were subtracted from the buffer-only spectrum then merged and analyzed in Primus 23 . 1D-scattering of different concentrations were merged, where appropriate, given that the lower concentrations better represent low q data points. The radius of gyration was calculated by ScÅtter 24 . The I 0 -values were calculated considering bovine serum albumin (BSA) as standard, where the R g was 33.6 ± 0.4 Å and I 0 93.13 ± 0.1. P(r) distance distribution functions were calculated and refined by the program ScÅtter. The distance r , where the P ( r ) functions approach zero probability, identifies the maximal dimension ( D max ) of the macromolecule. Using the refined data from ScÅtter in GNOM-format ab initio models for the corresponding SAXS-derived electron densities were calculated by averaging 20 rounds of DENSS 25 . These electron densities were fitted with structures and finally visualized by ChimeraX. The SAXS data sets are deposited in SASBDB 26 with accession codes SASDUW2. Hydrogen/deuterium exchange mass spectrometry (HDX-MS). Investigated proteins (ArnA, ArnB, ArnB ~ P, ArnA/ArnB ~ P) were employed as 50 µM concentrated stocks solutions in 20 mM Tris-Cl pH 8.0, 150 mM NaCl. From these, HDX reactions were prepared by an autosampler (LEAP Technologies) as follows: 6.5 µL of protein stock solution were pre-dispensed in a 96-well plate, 58.5 µL of a buffer (20 mM Tris-Cl pH 8.0, 150 mM NaCl) prepared with 99.9% D 2 O added and incubated at 25°C for 10/30/100/1,000/10,000 s. The HDX reaction was stopped by transferring 55 µL of the reaction to 55 µL of quench solution (400 mM KH 2 PO 4 /H 3 PO 4 , 2 M guanidine-HCl (pH 2.2) pre-dispensed in another 96-well plate cooled down to 1°C. 95 µL of the quenched reaction were injected into an ACQUITY UPLC M-Class system with HDX Technology (Waters) operating at 0.5°C through a 50-µL injection loop. Non-deuterated samples were generated analogously with an H 2 O-based buffer. Samples were washed out of the loop with water + 0.1% (v/v) formic acid (100 µL/min) and digested at 12°C in a cartridge (2 cm x 2 mm) filled with either bead-immobilized porcine pepsin or a 1:1 mixture of bead-immobilized protease type XVIII from Rhizopus sp. and protease type XIII from Aspergillus saitoi . The resulting peptides were collected on a trap column (2 mm x 2 cm; 0.5°C) filled with POROS 20 R2 reversed phase resin (ThermoFisher Scientific). After 3 min of digestion and trapping, the trap was placed in line with an ACQUITY UPLC BEH C18 1.7 µm 1.0 x 100 mm column (Waters) operating at 0.5°C and the peptides eluted with a gradient of water + 0.1% (v/v) formic acid (eluent A) and acetonitrile + 0.1% (v/v) formic acid (eluent B) at 30 µL/min, as follows: 0–7 min/95 − 65% A, 7–8 min/65 − 15% A, 8–10 min/15% A. The peptides were guided to a Synapt G2-Si mass spectrometer (Waters) and ionized by electrospray ionization (capillary temperature: 250°C; spray voltage: 3.0 kV). Mass spectra were acquired with MassLynX MS 4.1 (Waters) over 50 to 2,000 m/z in enhanced high-definition MS (HDMS E ) 27 , 28 or high-definition MS (HDMS) mode for non-deuterated and deuterated samples, respectively. A short spray of [Glu1]-fibrinopeptide B standard (Waters) every 45 s was employed for lock-mass correction. During peptide separation, the protease column was washed three times with 80 µL of 0.5 M guanidine-HCl in 4% (v/v) acetonitrile, and blank runs (double-distilled H 2 O) were performed between each sample. All measurements were performed in triplicates for each protease column type (separate HDX reactions). Peptides were identified ProteinLynx Global SERVER 3.0.1 (PLGS, Waters) and DynamX 3.0 (both Waters) from the non-deuterated samples acquired with HDMS E as described previously 29 employing low-energy, elevated-energy and intensity thresholds of 300, 100 and 1,000 counts, respectively and matched using a database containing the amino acid sequences of ArnA, ArnB, porcine pepsin, and their reversed sequences (peptide tolerance = automatic; fragment tolerance = automatic; min fragment ion matches per peptide = 1; min fragment ion matches per protein = 7; min peptide matches per protein = 3; maximum hits to return = 20; maximum protein mass = 250,000; primary digest reagent = non-specific; missed cleavages = 0; false discovery rate = 100). For quantification of deuterium incorporation with DynamX, peptides had to fulfil the following criteria: identification in two-thirds of the non-deuterated samples; minimum intensity of 30,000 counts; maximum length of 30 residues; minimum number of products of three and 0.1 products per residue; maximum mass error of 25 ppm; retention time tolerance of 0.5 minutes. Hereby, the datasets generated with porcine pepsin or after digestion with proteases type XIII and XVIII were pooled, and all spectra manually inspected. Residue-specific deuterium uptake of peptides was calculated with DynamX. If a residue was covered by a single peptide, the residue-specific deuterium uptake was equal to that of the whole peptide. In the case of overlapping peptides for a given residue, the residue-specific deuterium uptake was determined by the shortest peptide covering that residue. When multiple peptides were of the shortest length, the peptide with the residue closest to the C-terminus was utilized. Proteomics data evaluation Sample preparation and measuring of the S. acidocaldarius proteomes was performed as previously described 12 in collaboration with the bioanalytics MarMass facility of the Philipps University of Marburg. The timsTOF data was analyzed utilizing MaxQuant 2.1.3. Sequencing information for S. acidocaldarius DSM 639, comprising 2,222 entries, was retrieved from the UniProt database. For label-free quantification (LFQ) analysis, MaxQuant parameters were configured as follows: maximum peptide mass of 4,000 Da, allowance of up to three modifications per peptide, up to three missed cleavages, and the necessity of MS/MS for LFQ comparisons; all other parameters remained at default settings. Protein abundances from biological triplicates of the ΔArnA, ΔArnB and the wild type strain were filtered using a two-tailed student’s t-test (p 0.5 log 2 fold-change. Visualization was done using the pheatmap R package 30 in addition to the gplots package 31 . The volcano plots were constructed by plotting the log 2 fold-change in protein abundance on the x-axis against the negative logarithm (base 10) of the p-values (calculated from the student’s t-test) on the ordinate. Plots were created using the ggplot2 32 and ggrepel 33 packages in R, providing a comprehensive visualization of the proteomic changes in the context of significance levels. Gene Ontology (GO) enrichment analysis was performed on the sets of significant proteins (p < 0.05) from each comparison using GOATOOLS library version 1.2.3 34 . The significance of the GO enrichment analysis was determined by applying the Benjamini-Hochberg procedure with a false discovery rate (FDR) correction at a significance threshold of p = 0.05. The analysis was carried out with gene ontology terms specific to S. acidocaldarius DSM639, obtained via the QuickGO 35 annotation tool. Mutagenesis of ArnB threonines For point mutation of the different ArnB mutants 10–60 ng of starting vector was mixed with 150 ng of corresponding primers (Table S1 ), 1.5 µL DMSO, 1 µL dNTP mix (NEB), 0.5 µL of Phusion DNA-polymerase stock (~ 6 mg/mL) and adjusted to 50 µL with H 2 O. The PCR was performed for 18 cycles, primer suitable annealing temperatures (50–56°C) and an elongation temperature of 72°C. PCR was followed by a DpnI (2 µL, NEB) digest (37°C, 2h) and transformation into E. coli DH5α cells. Afterwards plasmids were extracted from overnight cultures using the QIAprep Spin Miniprep Kit (Quiagen) and checked by Sanger sequencing. Declarations Author Contributions: L.K.: Conceptualization, Formal analysis, Investigation, Methodology, Project administration, Validation, Visualization, Supervision of L.S., Writing – original draft, and Writing – review and editing; W.S.: Investigation of HDX experiments, Formal analysis of HDX data, Writing – original draft, and Writing – review and editing; M.W.: Formal analysis of MS data and Visualization; F.B.: Investigation, and Supervision of A.P.; M.S.V.: Investigation, and Project administration; L.S.: Investigation; A.P.: Investigation; S.H.: Investigation of SAXS experiments, and Formal analysis of SAXS data; S.V.A.: Funding acquisition, Project administration, Supervision, Writing – original draft, and Writing – review and editing; L.O.E.: Conceptualization, Data curation, Formal analysis, Funding acquisition, Project administration, Resources, Supervision, Writing – original draft, and Writing – review and editing. Competing Interest Statement: The authors declare no competing interests. Classification: Biological Sciences / Biochemistry. Data availability All data can be found in the supplementary information text or excel files. Protein structures have been uploaded to the PDB. Acknowledgements We thank the beamline staff of the Swiss Light Source (SLS), PSI, Villigen, Switzerland, as well as the beamline staff of the ESRF, Grenoble, France, for their support. We also want to thank the staff of Marburg Mass spectrometry and crystallization facility (MarXtal) for technical support. We acknowledge support by the German Research Council (DFG) through the core facility for HDX-MS (project numbers 260989694 and 324652314 to Gert Bange, Marburg). We want to thank the Life? program of the Volkswagen Foundation for funding (Grant: Az-96727). References Jarrell, K. F. & Albers, S. V. The archaellum: an old motility structure with a new name. Trends Microbiol. 20 , 307–312 (2012). Hoffmann, L. et al. Expanding the archaellum regulatory network – the eukaryotic protein kinases ArnC and ArnD influence motility of Sulfolobus acidocaldarius. Microbiologyopen 6 , 1–14 (2016). Lassak, K. et al. Molecular analysis of the crenarchaeal flagellum. Mol. Microbiol. 83 , 110–124 (2012). Lassak, K., Peeters, E., Wróbel, S. & Albers, S. V. The one-component system ArnR: a membrane-bound activator of the crenarchaeal archaellum. Mol. Microbiol. 88 , 125–139 (2013). Hoffmann, L. et al. Structure and interactions of the archaeal motility repression module ArnA-ArnB that modulates archaellum gene expression in Sulfolobus acidocaldarius. J. Biol. Chem. 294 , 7460–7471 (2019). Reimann, J. et al. Regulation of archaella expression by the FHA and von Willebrand domain-containing proteins ArnA and ArnB in Sulfolobus acidocaldarius. Mol. Microbiol. 86 , 24–36 (2012). Ye, X. et al. The Phosphatase PP2A Interacts With ArnA and ArnB to Regulate the Oligomeric State and the Stability of the ArnA/B Complex. 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The FHA domain protein ArnA functions as a global DNA damage response repressor in the hyperthermophilic archaeon Saccharolobus islandicus. MBio 14 , e0094223 (2023). Ellen, A. F. et al. Proteomic analysis of secreted membrane vesicles of archaeal Sulfolobus species reveals the presence of endosome sorting complex components. Extremophiles 13 , 67–79 (2009). Kumar, A. et al. Phosphorylation-induced unfolding regulates p19INK4d during the human cell cycle. Proc. Natl. Acad. Sci. U. S. A. 115 , 3344–3349 (2018). Querfurth, C. et al. Circadian conformational change of the Neurospora clock protein FREQUENCY triggered by clustered hyperphosphorylation of a basic domain. Mol. Cell 43 , 713–722 (2011). Thapar, R. Structural Basis for Regulation of RNA-Binding Proteinsby Phosphorylation. ACS Chem. Biol. 10 , 652 (2015). Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. Sect. D Biol. Crystallogr. 66 , 486–501 (2010). Afonine, P. V. et al. Towards automated crystallographic structure refinement with phenix.refine. Acta Cryst. 68 , 352–367 (2012). Pernot, P. et al. Upgraded ESRF BM29 beamline for SAXS on macromolecules in solution. J. Synchrotron Radiat. 20 , 660–664 (2013). Petoukhov, M. V. et al. New developments in the ATSAS program package for small-angle scattering data analysis. J. Appl. Crystallogr. 45 , 342–350 (2012). Konarev, P. V., Volkov, V. V., Sokolova, A. V., Koch, M. H. J. & Svergun, D. I. PRIMUS: a Windows PC-based system for small-angle scattering data analysis. J. Appl. Crystallogr. 36 , 1277–1282 (2003). Tully, M. D., Tarbouriech, N., Rambo, R. P. & Hutin, S. Analysis of SEC-SAXS data via EFA deconvolution and Scatter. J. Vis. Exp. 2021 , (2021). Grant, T. D. Ab initio electron density determination directly from solution scattering data. Nat. Methods 2018 153 15 , 191–193 (2018). Valentini, E., Kikhney, A. G., Previtali, G., Jeffries, C. M. & Svergun, D. I. SASBDB, a repository for biological small-angle scattering data. Nucleic Acids Res. 43 , D357–D363 (2015). Geromanos, S. J. et al. The detection, correlation, and comparison of peptide precursor and product ions from data independent LC-MS with data dependant LC-MS/MS. Proteomics 9 , 1683–1695 (2009). Li, G. Z. et al. Database searching and accounting of multiplexed precursor and product ion spectra from the data independent analysis of simple and complex peptide mixtures. Proteomics 9 , 1696–1719 (2009). Joiner, J. D. et al. HilE represses the activity of the Salmonella virulence regulator HilD via a mechanism distinct from that of intestinal long-chain fatty acids. J. Biol. Chem. 299 , 105387 (2023). Kolde, R. CRAN - Package pheatmap. (2019). Warnes, G. R. et al. Various R Programming Tools for Plotting Data [R package gplots version 3.1.3]. (2022). Wickham, H. ggplot2: Elegant Graphics for Data Analysis. Springer (2016). Slowikowski, K. Automatically Position Non-Overlapping Text Labels with ‘ggplot2’ [R package ggrepel version 0.9.4]. (2023). Klopfenstein, D. V. et al. GOATOOLS: A Python library for Gene Ontology analyses. Sci. Rep. 8 , 1–17 (2018). Binns, D. et al. QuickGO: a web-based tool for Gene Ontology searching. Bioinformatics 25 , 3045 (2009). Additional Declarations There is NO Competing Interest. Supplementary Files 20240213SupportingInformationArnBsubmissionfile.docx SupplementaryDataset1HDXMS.xlsx Dataset 1 HDX-MS SupplementaryDataset2Proteomics.xlsx Dataset 2 Proteomics SupplementaryDataset3Foldseekanalysis.xlsx Dataset 3 Foldseek analysis 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. 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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-3955852","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":274087349,"identity":"19501a20-e4cc-47a9-a604-ee3b100aee7b","order_by":0,"name":"Lars-Oliver Essen","email":"data:image/png;base64,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","orcid":"https://orcid.org/0000-0003-4272-4026","institution":"Philipp University of Marburg","correspondingAuthor":true,"prefix":"","firstName":"Lars-Oliver","middleName":"","lastName":"Essen","suffix":""},{"id":274087350,"identity":"3fc9a1b6-f331-48ab-a816-bb010e16f909","order_by":1,"name":"Lukas Korf","email":"","orcid":"","institution":"Philipp University of Marburg","correspondingAuthor":false,"prefix":"","firstName":"Lukas","middleName":"","lastName":"Korf","suffix":""},{"id":274087351,"identity":"73a44ca9-9c00-456a-aebd-c441afa302e4","order_by":2,"name":"Wieland Steinchen","email":"","orcid":"","institution":"Philipps-University Marburg","correspondingAuthor":false,"prefix":"","firstName":"Wieland","middleName":"","lastName":"Steinchen","suffix":""},{"id":274087352,"identity":"a465c8a8-9405-4317-b1cb-2e96a562e524","order_by":3,"name":"Mohamed Watad","email":"","orcid":"","institution":"Philipp University of Marburg","correspondingAuthor":false,"prefix":"","firstName":"Mohamed","middleName":"","lastName":"Watad","suffix":""},{"id":274087353,"identity":"7e322f39-89cb-4c7d-8b4f-8f6183d1dfde","order_by":4,"name":"Filipp Bezold","email":"","orcid":"","institution":"Philipp University of Marburg","correspondingAuthor":false,"prefix":"","firstName":"Filipp","middleName":"","lastName":"Bezold","suffix":""},{"id":274087354,"identity":"f79fcb51-71e5-47db-a84a-e721fdb8de8c","order_by":5,"name":"Marian Vogt","email":"","orcid":"","institution":"Philipp University of Marburg","correspondingAuthor":false,"prefix":"","firstName":"Marian","middleName":"","lastName":"Vogt","suffix":""},{"id":274087355,"identity":"86c86b13-7e29-4927-bc10-96e6e04b1280","order_by":6,"name":"Leon Selbach","email":"","orcid":"","institution":"Philipp University of Marburg","correspondingAuthor":false,"prefix":"","firstName":"Leon","middleName":"","lastName":"Selbach","suffix":""},{"id":274087356,"identity":"27a4f433-7fca-49e6-ac6c-91a08d737aa1","order_by":7,"name":"Alexander Penner","email":"","orcid":"","institution":"Philipp University of Marburg","correspondingAuthor":false,"prefix":"","firstName":"Alexander","middleName":"","lastName":"Penner","suffix":""},{"id":274087357,"identity":"021bc2af-24db-47de-827b-724745de04be","order_by":8,"name":"Maxime Tourte","email":"","orcid":"","institution":"Albert-Ludwigs-University Freiburg","correspondingAuthor":false,"prefix":"","firstName":"Maxime","middleName":"","lastName":"Tourte","suffix":""},{"id":274087358,"identity":"68cde947-a472-4ad4-8582-7012e698da0c","order_by":9,"name":"Sebastian Hepp","email":"","orcid":"","institution":"Philipp University of Marburg","correspondingAuthor":false,"prefix":"","firstName":"Sebastian","middleName":"","lastName":"Hepp","suffix":""},{"id":274087359,"identity":"da9e1412-2aca-41c6-b155-8428322a4b30","order_by":10,"name":"Sonja-Verena Albers","email":"","orcid":"https://orcid.org/0000-0003-2459-2226","institution":"University of Freiburg","correspondingAuthor":false,"prefix":"","firstName":"Sonja-Verena","middleName":"","lastName":"Albers","suffix":""}],"badges":[],"createdAt":"2024-02-14 10:32:04","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3955852/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3955852/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":52405031,"identity":"4b24c1ca-6d9e-45cc-a23c-62167af833e4","added_by":"auto","created_at":"2024-03-11 08:39:08","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1226851,"visible":true,"origin":"","legend":"\u003cp\u003eOverall Structure of the ArnAB complex and comparison with \u003cem\u003eSa\u003c/em\u003evWA2. A: Full structure of ArnAB complex and domain overview showing the ArnA ZnF domain interaction site. Domains are color-coded as follows: β-sandwich (green), vWFA (pink/purple), C-terminal domain (blue), ZnF domain (yellow). B: Focus view on the ZnF interaction site with important residues showing as sticks.C: MIDAS site of the vWFA domain of ArnB reveals Na\u003csup\u003e+\u003c/sup\u003e interacting residues. D: ArnB and vWA2 (grey) superposition with secondary structure elements numbering. The magnification shows the C-terminal domain of ArnB with threonine residues shown as sticks and OH-groups as balls, structurally highlighting potential FHA interaction sites. E: \u003cem\u003eAb initio\u003c/em\u003e envelope as calculated from SAXS-derived scattering vectors using 20 iterations of DENSS\u003csup\u003e25\u003c/sup\u003e for electron density modeling and ScÅtter\u003csup\u003e24\u003c/sup\u003e for refining GNOM-processed SAXS data. For the ArnAB complex (top) a D\u003csub\u003emax\u003c/sub\u003e value of 15.7 nm was derived (R\u003csub\u003eg\u003c/sub\u003e 4.16 nm), the DENSS model density has a resolution of 33 Å; the values for ArnB (bottom) alone are D\u003csub\u003emax\u003c/sub\u003e=7.5 nm, Rg=2.41 nm, resolution=30 Å. Structural models for the FHA domain of ArnA (orange), ArnB (grey) and the ArnB/ArnA-ZnF complex were fitted by ChimeraX into SAXS densities.\u003cbr\u003e\n\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-3955852/v1/91ab23beb7d846f76587b7e8.png"},{"id":52405176,"identity":"c2a2ae87-a0d4-4cd5-826c-a5ccfb5e4d02","added_by":"auto","created_at":"2024-03-11 08:55:08","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":818965,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePromiscuity of ArnB phosphorylation sites. A:\u003c/strong\u003e Sequence view of ArnB with trypsin cleavage sites, amino acid count and highlighted all potential phosphorylation sites found during this study. Domain coloring is analogous to Figure 1. \u003cstrong\u003eB:\u003c/strong\u003e Impact of phosphorylation time on threonine phosphorylation by MS. Green highlights newly found threonines at the respective time point. \u003cstrong\u003eC/D:\u003c/strong\u003e SDS gel pulldown assays showing that phosphorylation time has an impact on ArnB mutants, as different ArnB mutants (upper bands) are not able to sufficiently interact with ArnA (lower bands) when phosphorylation time is limited and, thus, cannot be pulled down anymore.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-3955852/v1/cda5ab21a16bff19d30c0508.png"},{"id":52405038,"identity":"b4aa7f9b-123e-4122-b981-1494250f0aa8","added_by":"auto","created_at":"2024-03-11 08:39:08","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":404267,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHDX reveals ArnA ArnB interaction sites and impact of phosphorylation.\u003c/strong\u003e \u003cstrong\u003eA:\u003c/strong\u003e Cumulative HDX, i.e. highest scoring change per any given time point, of the phosphorylated ArnAB where the phosphorylated ArnB HDX is subtracted from, revealing the interaction sites of ArnB with ArnA. The dashed box focuses on the FHA interaction with the C-terminal helix as a possible interaction site. The legend is representative for the whole figure. B: Impact of phosphorylation on ArnB as shown by cumulative HDX of ArnB-phosphorylated ArnB. The changes in HDX suggest a structure wide impact of the phosphorylation.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-3955852/v1/40a3f20ea2def3e63a39108f.png"},{"id":52405245,"identity":"2d7c2edf-1537-4e58-9211-20c44cd58fc0","added_by":"auto","created_at":"2024-03-11 09:03:08","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":433043,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eProteomics analysis of Sulfolobus acidocaldarius ΔarnA and ΔarnB strains in nutrient rich and starved conditions compared to the wild type. A/B: \u003c/strong\u003eArnA/B knockout proteomic analysis visualized as volcano plots in respect to the corresponding WT nutritional state. The top plots show nutrient rich conditions and the bottom plots nutrient starved conditions. Sample size comprises n=3 biological triplicates per sample, including respective WT samples, as well as two technical duplicates per measurements. Protein levels that have p-value \u0026lt; 0.05 and a log\u003csub\u003e2\u003c/sub\u003e Fold Change of \u0026gt;0.5 (i.e. \u0026gt;50%) are colored blue, i.e. down regulated in the respective knockout and nutritional state, or red, i.e. up regulated in the respective knockout and nutritional state. \u003cstrong\u003eC/D:\u003c/strong\u003e Gene enrichment analysis of respective protein hits, clustering them into GO terms revealing mostly metabolic involvement. \u003cstrong\u003eE/F:\u003c/strong\u003e Scatter plots of proteins found in both ArnA and ArnB knockout reveal high cross-effected protein rate, highlighting their cooperative role in S. acidocaldarius. \u003cstrong\u003eG:\u003c/strong\u003e Venn diagrams of ArnA and ArnB knockouts show that only ~half of the affected proteins (p\u0026lt;0.05) can be found in both knockouts, leaving room for individual roles.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-3955852/v1/24ace09356edb98d6cf7e250.png"},{"id":52405138,"identity":"44cfee16-cd23-4115-97e4-a04299570329","added_by":"auto","created_at":"2024-03-11 08:47:08","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1167432,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eArnB is a structural homolog of the COPII main membrane curvature factor Sec23/24. A:\u003c/strong\u003e Sec23/24 complex with analogous domain coloring to ArnB but in paler colors. Accordingly, the Sec23/24 helical domain represents the C-terminal domain of ArnB and the C-terminal Sec23/24 domain is colored orange. \u003cstrong\u003eB:\u003c/strong\u003e Superposition of Sec23/24 with ArnB without the C-terminal domain of Sec23/24 for clarification reasons, showing high structural alignment. \u003cstrong\u003eC:\u003c/strong\u003eSuperposition of ArnA FHA domain onto the C-terminal domain of Sec23/24 also reveals structural similarities and aligns well with a potential interacting position in a respective ArnAB complex.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-3955852/v1/df90a6cb8de6dec329fb52a8.png"},{"id":52405032,"identity":"57ccdc6c-6caf-46cc-884c-a9c7c91128e7","added_by":"auto","created_at":"2024-03-11 08:39:08","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":679879,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRole of heterodimerization to the ArnAB complex in different functional scenarios.\u003c/strong\u003e The phosphorylation-dependent formation of tight ArnAB complexes due to tethering via ZnF domains and FHA-pThr interaction apparently plays a role in the archaellum regulatory network and several metabolic pathways. A ‘loose’ ArnAB complex is formed by the 1:1 interaction between the ArnA-ZnF domain and ArnB. This single tether is augmented by ArnC-dependent phosphorylation of the ArnB_C domain and the ArnA-FHA domain (tight ArnAB complex). Double-tethered ArnAB complexes may exist as stoichiometric 1:1 assemblies (top, right) or multimeric higher complexes prone to polymerization (bottom, right). In the tight ArnAB complex and the ArnA's long linker region may enable binding of further interaction partners (mid, right). In this context, transition to loose ArnAB complexes and disassembly is driven by dephosphorylation due to PP2A triggering further arlB production.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-3955852/v1/6d9fdc3caf255ed083b8851e.png"},{"id":52405396,"identity":"b30f82b9-740d-4946-9de5-d99a08cdb2cb","added_by":"auto","created_at":"2024-03-11 09:11:14","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4804806,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3955852/v1/037c887d-b127-4173-9ddf-d8e20a82eeb0.pdf"},{"id":52405040,"identity":"8716a366-b678-4849-9706-da1a05ac0326","added_by":"auto","created_at":"2024-03-11 08:39:08","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":6134385,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"20240213SupportingInformationArnBsubmissionfile.docx","url":"https://assets-eu.researchsquare.com/files/rs-3955852/v1/d73d3fad97bd1e9d660f4c59.docx"},{"id":52405179,"identity":"91ddf30d-7433-4a01-99ee-b00cda3311b5","added_by":"auto","created_at":"2024-03-11 08:55:08","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":583327,"visible":true,"origin":"","legend":"\u003cp\u003eDataset 1 HDX-MS\u003c/p\u003e","description":"","filename":"SupplementaryDataset1HDXMS.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-3955852/v1/76c25efdcd0b7f6b38dbbc77.xlsx"},{"id":52405041,"identity":"571772ae-1abc-4d2d-86b0-c348488ff6e1","added_by":"auto","created_at":"2024-03-11 08:39:08","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":3461898,"visible":true,"origin":"","legend":"\u003cp\u003eDataset 2 Proteomics\u003c/p\u003e","description":"","filename":"SupplementaryDataset2Proteomics.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-3955852/v1/8a3f18e73183c65a1ee83bc2.xlsx"},{"id":52405393,"identity":"0e3f7ad6-fd11-445d-809b-02887369bd2a","added_by":"auto","created_at":"2024-03-11 09:11:08","extension":"xlsx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":170849,"visible":true,"origin":"","legend":"\u003cp\u003eDataset 3 Foldseek analysis\u003c/p\u003e","description":"","filename":"SupplementaryDataset3Foldseekanalysis.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-3955852/v1/7273419e243be00417e5a251.xlsx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Sequential conformational transition of ArnB, an archaeal ortholog with Sec23/Sec24 core motif","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe archaellum serves as the primary motility apparatus in Archaea, facilitating self-locomotion and enabling adaptive habitat transitions \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Its responsiveness to diverse environmental stimuli is governed by a regulatory network known as the archaellum regulatory network (Arn) \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Within this intricate system, which regulates transcriptional and translational adaptations, the proteins ArnA and ArnB play pivotal roles in the Crenarchaeon \u003cem\u003eSulfolobus acidocaldarius\u003c/em\u003e, particularly in the regulation of \u003cem\u003earlB\u003c/em\u003e (formerly \u003cem\u003eflaB\u003c/em\u003e), the gene encoding the archaellin \u003csup\u003e\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. In the context of nutrient availability in \u003cem\u003eS. acidocaldarius\u003c/em\u003e, ArnA and ArnB orchestrate the regulation of \u003cem\u003earlB\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Notably, the von Willebrand factor domain-containing ArnB forms a complex with the forkhead-associated domain (FHA) protein ArnA \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Deprivation of either ArnA or ArnB results in an upregulation of \u003cem\u003earlB\u003c/em\u003e expression, leading to hypermotility in the organism \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Furthermore, the interplay between nutrient levels and the formation of the ArnAB complex affecting the formation of archaella has been elucidated \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Starvation conditions induce a loss of interaction within the complex, subsequently promoting the expression of \u003cem\u003earlB\u003c/em\u003e. Consequently, ArnA and ArnB are identified as negative regulators of the archaellum when complex formation is established. Phosphorylation-dependent interactions between ArnA and ArnB are facilitated by the phosphatase PP2A and the kinase ArnC, maintaining a delicate balance between complex formation (phosphorylation) and dissociation (dephosphorylation) \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Despite in-depth \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e studies, the exact interaction mode of ArnA and ArnB, purportedly located in the C-terminal region of ArnB, remains elusive. Moreover, the mechanism by which the ArnAB complex controls the ArlB levels remains enigmatic. The formation of ArnAB oligomers that depends on phosphorylation is another feature \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Intriguingly, we found that the structural characteristics of ArnB with its intriguingly associated β-sandwich, von Willebrand factor type A (vWFA) and α-helical domains form a structural blueprint widely distributed among all domains of life, which also includes the eukaryotic COPII-vesicle assembly factors Sec23/Sec24. However, functional divergence as observed in archaeal proteins like the histones \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e, which are functionally distinct in eukaryotes despite sharing a common core fold, illustrates the limitation of relying solely on structure-based function inference. Given this caveat we performed a biochemical and structural analysis of ArnAB interaction that revealed a novel mode of sequential conformational change of its Sec23/Sec24 core motif by phosphorylation.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eOverall structure of the ArnA-ArnB complex and structural comparison with\u003c/span\u003e \u003cspan type=\"ItalicUnderline\" class=\"ItalicUnderline\" name=\"Emphasis\"\u003eSa\u003c/span\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003evWA2\u003c/span\u003e\u003c/p\u003e \u003cp\u003eWe were able to solve the crystal structure of the vWA2 paralog \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e and archaellum regulating factor ArnB (UniProt: Q4J9H3, saci_1211) in its complex with ArnA (UniProt: Q4J9H4, saci_1210). The ArnAB cocrystals comprise two complexes per asymmetric symmetry unit, whose structures were solved by molecular replacement and refined at 2.5 \u0026Aring; resolution. The ArnAB complexes are defined by electron density for residues T2-S380 of ArnB and the zinc-finger (ZnF) domain of ArnA (P16-K42, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, PDB: 8S05). The latter implies a loss of the FHA domain and the linker region of ArnA, possibly by unspecific proteolysis as observed before when solving the structure of the ArnA FHA domain \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. The overall architecture of ArnB corresponds mostly to its paralog, the van Willebrandt Factor A (vWFA)-containing protein \u003cem\u003eSa\u003c/em\u003evWA2, including the vWFA domain, an eight-stranded β-sandwich whose topology is split by the vWFA domain, as well as the C-terminal, four helix bundle motif, that has been classified as ArnB_C domain (InterPro entry IPR040929) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA) \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. In contrast to \u003cem\u003eSa\u003c/em\u003evWA2, the ArnB_C domain of ArnB harbors an elongated helix pair at the terminal region of the motif, revealing one of the most substantial structural differences between ArnB and \u003cem\u003eSa\u003c/em\u003evWA2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). This extension presents additional threonine residues that allow for potential interaction of the forkhead-associated (FHA) domain of ArnA, known for its ability to bind phospho-threonines, with ArnB (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD right panel) \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Moreover, ArnB features an additional helix (P95-Q103) in the vWFA domain between β5 and α2 of \u003cem\u003eSa\u003c/em\u003evWA2 increasing the total helix count to ten (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). The vWFA domain of ArnB also harbors a Na\u003csup\u003e+\u003c/sup\u003e ion coordinated by D46, S50, T110, T135 and D136 in the metal ion-dependent adhesion site (MIDAS, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWhile ArnA interacts strongly with its FHA domain to phosphorylated ArnB (see below), the zinc finger domain (ZnF domain) itself is sufficient to promote an interaction without a post-translational modification of ArnB. This ZnF domain belongs to the RanBP2-type (IPR001876) and is characterized as a ZnF ribbon domains by two consecutive, distorted β-hairpin motifs, which together a zinc ion via C21, C24, C35 and C38. The interface of the ZnF domain of ArnA with ArnB has a rather moderate size of 504/574 \u0026Aring;\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e for ArnAB chains A/C and B/D, respectively. The interactions are mostly of hydrophobic nature and include the C-terminal β-hairpin motif (D31-Q41) of the ZnF domain as well as the N-terminal β1-β2 loop (H12-K21) of the β-sandwich domain, it\u0026rsquo;s α-helical linker to the ArnB_C domain (V286-I293) and adjacent residues of the ArnB_C domain facing the ZnF domain. Accordingly, the ArnA-ArnB interaction based on the ZnF domain appears to be rather weak as indicated by pulldown assays (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA), but traceable by mass photometry \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. A further point for the uniqueness of the ZnF-mediated ArnA-ArnB interactions are AF2-multimodels, which were unbiased of the ArnAB structure and show almost an identical interaction as in the ArnA-ArnB cocrystal structure for 4 of the 5 predicted ArnA-ArnB models (Figure S2) with displacement r.m.s.d. values of 1.57\u0026ndash;1.65 \u0026Aring; for M1-Q25 of the ZnF domain. This indicates that the intrinsic sequence covariation for ArnA and ArnB domains is already significant enough to provide a robust indicator for the relatively small ArnB/ZnF domain interface. Moreover, an ArnA-ArnB interaction is also displayed in solution by SAXS data (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE) as the \u003cem\u003eab initio\u003c/em\u003e envelope as derived from the SAXS data is fittable to the ArnA-FHA and ArnAB crystal structures. Here, the pair distance distribution function P(r) suggests an overall elongated shape (Figure S3) and thereby supports an ArnA-ArnB interaction based on the ZnF domain, as in the crystal structure, with a flexible region followed by an unbound FHA domain due to a lack of pThr anchor points.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePromiscuity of ArnB phosphorylation-dependent interaction sites\u003c/h2\u003e \u003cp\u003eIn the search for the entire interaction site of the strong ArnA-ArnB interaction based on phospho-threonine interaction, we performed a comprehensive mass spectrometry-based analysis. Notably, there are many potential interaction sites found in ArnB, especially in the C-terminal HTH motif (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA) where interaction appears to be most likely, based on structural analysis. Accordingly, we first opted to investigate the phosphorylation pattern by the kinase ArnC (UniProt: Q4J9J0, Saci_1193) with the previously reported phosphorylation conditions \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Interestingly, we found that many threonines in the ArnB_C domain were phosphorylated during our \u003cem\u003ein vitro\u003c/em\u003e phosphorylation, raising some interesting questions about the native phosphorylation conditions. However, as 60 minutes of incubation time at 55\u0026deg;C yielded many different potential interaction sites we opted to find the ones that are phosphorylated first. Hence, we investigated the effect of different incubation times on the phosphorylation pattern of ArnB. In time points of 5 min, 15 min, 30 min and 60 min (resembling our reference sample) the phosphorylation pattern of ArnB was analyzed in a tryptic digest MS experiment. Interestingly, ArnA could still be efficiently pulled down along with ArnB during purification with phosphorylation time of little as 5 min. Moreover, phosphorylation of threonines located closer to the N-terminal side of the ArnB_C domain takes place only after T353, T354, T359, T363 and T375 are phosphorylated and only until T322, which aligns with the known phenomenon that multiple phosphorylation usually occur as clusters in a protein \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Accordingly, these main threonines are phosphorylated after 5 minutes already, while other phosphorylated threonines that have potential counterparts in vWA2 are found phosphorylated only after 15 minutes or more incubation time. This may indicate ordered processivity of hyperphosphorylation, like in the cyclin\u0026ndash;Cdk1\u0026ndash;Cks1 system \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e and goes along with the threonines being located in the extension of the C-terminal HTH motif of ArnB, besides T375, which are therefore not present in vWA2. Additionally, it is reported that vWA2 does not interact with ArnA \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e, supporting the assumption that the C-terminal HTH extension is the main interaction side for the ArnA FHA domain.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAs this narrowed down the possible interaction sites, we attempted to identify the exact position of the ArnB-FHA interaction by a comprehensive alanine mutagenesis study. Inducing the mutation of either T353A\u0026thinsp;+\u0026thinsp;T354A or T359A\u0026thinsp;+\u0026thinsp;T363A it was not possible for the FHA domain to interact with ArnB anymore, if phosphorylated for 5 min or less. However, increasing the phosphorylation time to 60 min, ArnA could be pulled down with ArnB without issues again. Additionally, even when threonines that were found to be phosphorylated only after 5 min were mutated as well, ArnA could still be pulled down along with ArnB, represented by the ArnB-T343A-T344A-T353A-T354A-T359A-T363A-T371A-T375A mutant (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eB). In general, this gives an idea of the more likely interaction side of the FHA domains location at the C-terminal elongation of the ArnB_C domain, but leaves it open if multiple FHA interaction sites play a role in the interaction of ArnA and ArnB. However, the hyper-phosphorylation we observed leads to multiple structural rearrangements, as discussed in the following, and hence are not necessarily interaction sites but structural factors important for a correct ArnB-FHA interaction.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eHDX-MS reveals phosphorylation-dependent structural relieves of ArnB\u003c/h2\u003e \u003cp\u003eIn order to get insights into the interaction site(s) between ArnA and phosphorylated ArnB in solution, we made use of the property of amide protons to exchange for protons from the aqueous solvent, the extent of which was traced upon incubation of the proteins in deuterated buffer. This hydrogen/deuterium exchange (HDX) was then, after digestion of the proteins into peptides, quantified by mass spectrometry (MS).\u003c/p\u003e \u003cp\u003eWe subjected ArnA, ArnB, phosphorylated ArnB, and the ArnA/phosphorylated ArnB complex to HDX-MS experiments, allowing us to resolve, \u003cem\u003ei)\u003c/em\u003e the conformational changes that ArnB undergoes upon phosphorylation, and \u003cem\u003eii)\u003c/em\u003e the regions of ArnA and phosphorylated ArnB establishing the interaction interface in their complex.\u003c/p\u003e \u003cp\u003eFor ArnA and ArnB, we could identify 80 and 145 peptides, respectively, that covered more than 90% of their corresponding amino acid sequences (Figures. S1A and S2A, Supplementary Dataset 1). The HDX profile of ArnA corroborated its predicted domain topology, in particular the disordered nature of the linker (S28-N99, where maximal HDX was reached after 10 s of deuteration) joining the N-terminal ZnF domain to the C-terminal FHA domain (Figure S4B). Upon complex formation of ArnA with phosphorylated ArnB, HDX reduction became apparent in both the ZnF domain and FHA domains thus marking the major sites of interaction for ArnA (S1C-D). Specifically, the phosphate recognition module of the FHA domain constituted by R132 and R147 exhibited the strongest HDX reduction consistent with their role in FHA-mediated phosphate recognition for strengthening the ArnA/ArnB interaction \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe HDX profile of ArnB itself primarily showed regions of higher-order structure (low HDX at 10 s of deuteration and progression in HDX over the time-course) and only small disordered areas coinciding with short linkers of the crystal structure (Figures S2B, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). However, phosphorylation of ArnB induces widespread conformational changes, according to HDX increases of the ArnB domain, in parts of the β-sandwich and vWFA domains vicinity (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). These changes may reflect either a partial unfolding event or a disentanglement of the three associated domains. Furthermore, binding of ArnA to phosphorylated ArnB, in turn, reduced the observed HDX rates in proximity of the ArnA-ZnF domain binding site (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Notably, ArnB residues D295-A310, constituting the N-terminal portion of helix α7, incorporate more deuterium upon phosphorylation, whereas a reduction was apparent upon ArnA binding (Figure S6). It may be hence speculated that the phosphorylation-induced conformational change could be a prerequisite for tight ArnA binding. Likely due to the phosphorylation of the threonine side chains, no peptides covering the C-terminus\u0026rsquo; residues T353 onwards could be retrieved thus precluding further conclusions on this presumed ArnA-FHA domain interaction site by HDX-MS of ArnB (Figure S5A). Overall, HDX-MS corroborates the binding site of the ArnA-ZnF domain on ArnB observed in the complex structure (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) and provides evidence for a secondary interaction site established with the ArnA-FHA phosphate recognition module.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003etims-ToF proteomics data for ArnA and ArnB deletion strains\u003c/h2\u003e \u003cp\u003eAlthough the roles of ArnA and ArnB in the archaellum regulatory network are established \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e, the mechanism by which regulation occurs under nutrient limitation remains undisclosed. Consequently, we analyzed the \u003cem\u003eSulfolobus acidocaldarius\u003c/em\u003e proteome of the \u003cem\u003eΔarnA\u003c/em\u003e and \u003cem\u003eΔarnB\u003c/em\u003e strains in two nutritional states in comparison with the respective wild type (WT). For this analysis biological triplicates were grown of each strain, and samples were taken under nutrient rich and starved conditions. Samples were measured in a timsTOF (trapped ion mobility spectrometer) mass spectrometer and quantified via label free quantification before averaging the technical duplicates of each sample and further analysis. These measurements led to the identification of 1,699-1,710 proteins per sample leading to the identification of 1,723 overall proteins of the 2,222 gene products known for the respective \u003cem\u003eSulfolobus acidocaldarius\u003c/em\u003e strain (Supplementary Dataset 2). The comparison of the knockout strains with their respective WT sample reveals 1,713-1,716 identified proteins per comparison with overlapping count of 1,694-1,704 (98.7\u0026ndash;99.4%). As the overall proteome is basically unaffected by the deletions, we investigated the effects of the deletion strains under higher stringency i.e. a two-tailed \u003cem\u003et\u003c/em\u003e-test with a p-value cutoff of \u0026lt;\u0026thinsp;0.05. This analysis revealed 325(rich)/384(starved) statistically changed proteins compared to the WT proteome for the ArnA knockout and 398(rich)/510(starved) for the ArnB knockout, respectively. In addition, we also included the stringency factor of a fold change of at least 50% to not only check for statistical relevance but also for biological effects (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA/B). As displayed in the volcano plot analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA/B, S8-9), this revealed 115(rich) / 243(starved) proteins for the \u003cem\u003eΔarnA\u003c/em\u003e and 185(rich) / 279(starved) proteins for the \u003cem\u003eΔarnB\u003c/em\u003e strain passing that high stringency test. This shows that under starvation conditions the effect on the proteome level is more prominent under nutrient starved conditions for both knockouts. Moreover, the effect of the ArnB knockout is also slightly more impactful on overall altered proteins both after t-testing and after employing the additional fold change cutoff of 50%. To see if these statistically relevant changes on the proteome level also reflect on the biology of \u003cem\u003eS. acidocaldarius\u003c/em\u003e, we conducted an intensive gene ontology (GO) term analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC/D). Interestingly, this analysis showed that both knockouts have a preference for metabolic enzymes, especially for additional nitrogen containing amino acids pathways. Nevertheless, purine/nucleotide, acetyl-CoA and carbohydrate metabolism enzymes were also significantly enriched after knockout of either ArnA or ArnB. Despite a small difference in statistical significance and actual enrichment score the GO term analysis delivered quite similar results for both knockouts. Notably, when comparing both knockouts with each other, only 43\u0026ndash;61% of the significantly altered proteins can be found in both deletion strains simultaneously (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG). However, those significantly altered proteins that can be found in both knockouts do share a very high correlation of 82\u0026ndash;86% (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE/F). Together this leads to the conclusion, that around half of the impact of knocking out either ArnA or ArnB is apparently based on the interaction of them with each other to some degree. \u003cem\u003eVice versa\u003c/em\u003e this means that the other half of the effected protein levels are apparently independent from the ArnA-ArnB interaction or at least without direct correlation. A comparison of these proteomics data with a deletion variant of \u003cem\u003eS. acidocaldarius\u003c/em\u003e that missed the GPN-loop GTPase \u003cem\u003eSa\u003c/em\u003eGPN and exhibited diminished motility \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e shows that the proteome changes of the hypermotile ArnA and/or ArnB deletion variants \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e are more modest. This suggests a more intimate involvement of ArnA and ArnB in the regulation of motility than of \u003cem\u003eSa\u003c/em\u003eGPN, which causes large-scale changes in the proteome network. Accordingly, there is no correlation for significantly altered protein levels between the \u003cem\u003eSa\u003c/em\u003eGPN knockout and the ArnA or ArnB knockouts (Figure S10). This suggests that the role of ArnA and ArnB in the archaeal regulatory network is independent of \u003cem\u003eSa\u003c/em\u003eGPN.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eEvolutionary context of ArnB, which contains the Sec23/Sec24-core motif\u003c/h2\u003e \u003cp\u003eIn the current state of literature, the function of ArnB, a homolog of the vWA2 protein in \u003cem\u003eS. acidocaldarius\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e, has been associated with the archaellum regulatory network. It has been reported that ArnB is a negative regulator of the archaellin arlB, regulated by the interaction with its partner ArnA. However, during investigation of ArnB we found structural similarities with the membrane curvature proteins Sec23/24, which are central components of the assembly machinery for eukaryotic COPII vesicles. Sec23/24 harbor a domain arrangement highly reminiscent of ArnB and even include an N-terminal ZnF domain like ArnA. Accordingly, Sec23/24 contain as a core motif the vWFA, β-sandwich and C-terminal helical domain, the latter being similar to the ArnB_C domain, besides an additional C-terminal domain (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Superposition of the Sec24 core motif (PDB: 1m2v, P301-I749 of chain B) and ArnB reveals a structural deviation of 5.7 \u0026Aring; for 302 Cα atoms. Here, differences between the Sec24 core motif and ArnB are mostly found for the length of helices in the helix pair α9/α10 of the ArnB_C domain and the relative orientation of the β-sandwich. Notably, the ZnF domain of Sec23/Sec24 and that of ArnA in the ArnAB complex pack to different sites of the β-sandwich of the core motif. In the ArnAB complex structure, the ZnF domain occupies a site made up by an edge between the β-sandwich and the ArnB_C domains, whereas in Sec23/Sec24 the ZnF-domain associates to the opposite site of the β-sandwich domain (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Another feature in this context is the packing between the Sec23/Sec24 core motif and the C-terminal domain. Although ArnB lacks the gelsolin-type C-terminal domain of Sec23/24, the FHA domain of ArnA is predicted to interact in a phosphorylation-dependent manner with the α-helix domain that resembles the packing of the Sec23/Sec24 motif and the C-terminal domain.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe close relationship between the ArnAB assembly and the eukaryotic Sec23/24 core motif prompt for a wider distribution of the Sec23/Sec24 core motif within the domains of life. Using foldseek and the ArnB crystal structure we found a wider occurrence for the ArnB/Sec23 core motif (Supplementary Dataset 3) than previously suggested by the structural relationship between vWA2 from \u003cem\u003eS. acidocaldarius\u003c/em\u003e and a vWA protein from the actinobacterium \u003cem\u003eCatenulispora acidiphila\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Structural orthologs are found in other bacterial phyla than the actinobacteria. For example, \u003cem\u003eEscherichia coli\u003c/em\u003e has a predicted structural ortholog, YfbK, whose domain topology is found in ~\u0026thinsp;5900 gene products (status 01/24, Figure S11A). Besides matching Alphafold2 models from Archaea including Euryarchaeota like \u003cem\u003eHalorubrum\u003c/em\u003e we also find Sec23/Sec24 core motifs in other \u003cem\u003eKorarchaeota\u003c/em\u003e and \u003cem\u003eHeimdallarchaeota\u003c/em\u003e (Figure S11B). More surprisingly, the closest structural hits outside the archaeal domain are found in the plant and fungal kingdoms despite marginal sequence identities. Orthologs in plants like \u003cem\u003eArabidopsis thaliana\u003c/em\u003e and \u003cem\u003eZea mays\u003c/em\u003e (Figure S11C) currently lack an assigned function. Notably, foldseek hits in the animal kingdom, apart from the expected Sec23/Sec24 orthologs, lack a direct structural relationship to the Sec23/Sec24 core motif and ArnB (Figure S11D).\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis work presents much needed structural information, supported in both solution and crystal structure, of the ArnAB complex and features more information about its \u003cem\u003ein vivo\u003c/em\u003e role. The role of ArnA, ArnB and its hetero complex has so far been associated with the archaellum regulatory network, hence the name Arn \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Nevertheless, their biological role is not yet fully understood leaving open questions of why they are found in exosomes and why they share a structural homology with the Sec23/24 complex. Recently, it was reported that the ArnA homolog from \u003cem\u003eSaccharolobus islandicus\u003c/em\u003e (\u003cem\u003eSis\u003c/em\u003e) interacts with promotor sequences supporting a transcription factor role for ArnA \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e, although DNA binding was rather unspecific. However, during our work, we could not find any support that ArnA from \u003cem\u003eS. acidocaldarius\u003c/em\u003e (\u003cem\u003eSa\u003c/em\u003eArnA) itself is capable of binding DNA. Moreover, there are no structural indications that either ArnB or ArnA have DNA binding sites, especially since the ZnF domain of ArnA, which would be an obvious target, is part of a protein-protein interface. As \u003cem\u003eSis\u003c/em\u003eArnA was purified from the native organism, its DNA binding activity may depend on additional interaction partners than ArnB or post-translational modifications, which require further elucidation.\u003c/p\u003e \u003cp\u003eFurthermore, our comprehensive proteomic data suggests involvement of ArnA and ArnB in anabolic metabolism pathways. Accordingly, knockout of either of them lead to a gene enrichment in enzymes concerning amino acid metabolism, especially N-rich amino acids, carbohydrates and acetyl-CoA metabolism. This is particularly resembled by changes in protein levels of the Gln-synthase (Q9HH09), succincyl-CoA ligase (Q4J9C0) or an MFS sugar transporter protein (Q4J7I0). Obviously, the known overproduction of the archaellum, an indicator of hypermotility, caused by the ArnA/ArnB knockouts \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e, indicates \u003cem\u003eper se\u003c/em\u003e changes of the metabolic repertoire in \u003cem\u003eS. acidocaldarius\u003c/em\u003e, when switching from a an immobile, sessile form to a planktonic lifestyle. Compared to other proteomic data regarding proteins that are involved in the Arn-system like the GPN-loop GTPase from \u003cem\u003eS. acidocaldarius Sa\u003c/em\u003eGPN, these changes are completely different \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. While only half of the statistically relevant proteome changes is common between ArnA and ArnB deletion strains, the intrinsic connection between both is also mirrored by the nearly symmetrical alterations observed in proteins that play pivotal roles in both strains. Therefore, we are confident to say that the metabolic impacts we observed are directly related to the specific knockouts. Accordingly, the large-scale proteome impacts observed for the \u003cem\u003eSa\u003c/em\u003eGPN knockout appear to be particularly more random. Notably, as \u003cem\u003eSa\u003c/em\u003eGPN is being considered part of the Arn system, physical association of the ArnAB complex with \u003cem\u003eSa\u003c/em\u003eGPN might link to a collaborative function of all three of them. This could also explain why the previously reported PP2A co-IP assay \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e pulls down not only ArnA and ArnB but \u003cem\u003eSa\u003c/em\u003eGPN as well. However, it may also be of coincidental nature and no physical association connects the ArnAB complex with \u003cem\u003eSa\u003c/em\u003eGPN. Nevertheless, the aspect of the ArnA-ArnB interaction corresponds to a \u0026lsquo;flip switch\u0026rsquo; depending on the phosphorylation state of ArnB and its respective oligomeric state. These states are regulated by the PP2A and ArnC phosphatase-kinase system and induce heterodimerization as well as higher oligomerization upon threonine phosphorylation based on the FHA-pThr interaction. Interestingly, the oligomerization processes possibly involve a (partially) unwinding or loosening of the ArnB motifs. Accordingly, the stand-alone domain of PP2A exhibits a more restricted array of control modes compared to ArnC, which exhibits an elongated N-terminal region containing a TPR-like helical domain in addition to its C-terminal Ser/Thr kinase domain and thereby offering a more diverse range of regulatory mechanisms.\u003c/p\u003e \u003cp\u003eThe Sec23/24-like domain architecture of ArnB with its β-sandwich, vWFA and C-terminal helical bundle domain is conserved among all domains of life. Captivatingly, the evolutionary connections between different kinds of proteins within all three domains of life is apparent in their structure with only moderate changes. However, due to a weak phylogenetic connection between them the functions of the eukaryotic Sec23/24 exert completely different functions than their archaeal predecessors. Therefore, the structural homology between ArnB features with Sec23/24 does not necessarily place ArnB with the COPII vesicle system, as structural homologs of ArnB can even be found in \u003cem\u003eEscherichia coli\u003c/em\u003e (Figure S11A). Nevertheless, two main scenarios appear likely: function in vesicle transport either arose late, i.e. in Asgard Archaea with their symbiotic lifestyles with bacteria, or upon the transition to Eukaryotes, i.e. during the manifestation of endosymbiosis. Moreover, a prerequisite for a functional switch may be the association with exosomes as observed before \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e, even when ArnA/ArnB are not directly involved in exosome formation.\u003c/p\u003e \u003cp\u003eInterestingly, ArnB demonstrates a phosphorylation-dependent alteration of its conformational flexibility as revealed by HDX-MS. Phosphorylation proceeds at multiple, but specific sites and follows a pattern consistent with a model in which ArnC-mediated phosphorylation starts at threonines located in the tip regions of the C-terminal helical bundle (T353-T363). Subsequently, it propagates to threonine sites, which are mostly buried within the helix bundle/vWFA domain interface (T371-T375), and finally extends to sites closer to the N-terminus of the helix bundle domain. It is worth noting that such a sequential phosphorylation and associated partial unfolding of the protein is not without precedence \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. For example, for p19\u003csup\u003eINK\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003ed\u003c/sup\u003e, an ankyrin-repeat protein, it is reported that the sequential phosphorylation within the helical repeat domain progressively destabilizes the N-terminal half of the protein, ultimately leading to the unfolding of the corresponding section \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Moreover, the casein kinase 1a (CK1a) of \u003cem\u003eNeurospora\u003c/em\u003e progressively hyper-phosphorylates the clock protein FREQUENCY (FRQ), which triggers a conformational change driven by clustered phosphorylation \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. In general, multi-phosphorylation exerts regulatory influence over protein structures, facilitating bidirectional transitions between ordered and disordered states \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Notably, the sequential part of the phosphorylation patterns apparently does not necessarily affect ArnB-FHA interaction, as different threonine mutants of ArnB were still able to bind the FHA domain of ArnA, as long as alternative phospho-sites were available (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC/D). However, these large-scale conformational changes observed in ArnB align with a dual-point attachment of ArnA facilitated by both its ZnF and FHA domains. This interaction could serve as the catalyst for the assembly of larger molecular complexes involving ArnA and ArnB, resembling but not identical to the established Sec23/Sec24 COPII coat formation.\u003c/p\u003e \u003cp\u003eMany of the intricacies surrounding the functionality and assembly of the ArnAB complex stem from uncertainties related to the oligomerization behavior, as well as the precise interaction point of the ArnA FHA domain and the role of the ArnA linker domain. The absence of structural support has led to various plausible scenarios. Consequently, we posit three primary operational modes governing the heterocomplex formation of ArnA and ArnB, based on our structurally supported findings. Firstly, these scenarios encompass the possibility of simple heterodimerization, where observed oligomers may lack biological relevance. This could be attributed to their potentially artificial nature \u003cem\u003ein vitro\u003c/em\u003e, stemming from a deficiency in regulated phosphorylation, underscored by the varying incubation times with ArnC, as illustrated in this and previous studies \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e top right). Secondly, it's conceivable that another element plays a role in ArnAB function. Both DNA and other proteins, such as \u003cem\u003eSa\u003c/em\u003eGPN and \u003cem\u003eSa\u003c/em\u003eUspA, identified in the previously reported co-IP assay on PP2A interaction partners, emerge as potential targets (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e mid right). Lastly, polymerization based on the FHA domain appears probable. Numerous phosphorylated states of the ArnAB complex \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e, especially with extended ArnC incubation times, hint at a multifaceted interplay facilitated by the FHA domain-pThr interaction, thereby mimicking a Sec23/24-like behavior (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e bottom right).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn conclusion, the intriguing phosphorylation-dependent interaction between the archaellum regulatory network proteins, ArnA and ArnB, not only adds to our understanding of archaellum-related functions but also positions them as potential precursors to COPII homologs. This suggests a broader significance, possibly making them among the earliest COPII-like proteins, which were already present in TACK Archaea.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eMaterials\u003c/h2\u003e \u003cp\u003eIf not stated otherwise chemicals and materials were obtained from Carl Roth. Primers were ordered from Microsynth.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eProtein expression\u003c/h2\u003e \u003cp\u003eOverexpression of recombinant proteins of this study was performed in LB medium (10 g/L tryptone, 10 g/L NaCl, 5 g/L yeast extract) supplemented with 34 µg/mL chloramphenicol, using BL21(DE3) Rosetta cells. After induction of gene expression (0.5 mM IPTG at OD\u003csub\u003e600nm\u003c/sub\u003e = 0.6–0.8) cultures were incubated overnight (16°C, 150 rpm, 18 h). Harvesting (20°C, 5,000 rpm, 20 min) was followed by resuspension in lysis buffer (150 mM NaCl, 50 mM Tris, pH = 8.0). Cell pellets were either used freshly for protein purification or frozen in liquid N\u003csub\u003e2\u003c/sub\u003e and stored at -80°C for later use.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eProtein purification without phosphorylation\u003c/h2\u003e \u003cp\u003eCell pellets were lysed using a micro-fluidizer (2 min/2L cell pellet equivalent) after harvesting. The lysate was centrifuged (18,000 rpm, 20°C, 20 min), the supernatant heat-treated (70°C, 20 min) and centrifuged again (18,000 rpm, 20°C, 20 min) before being loaded to a Ni-NTA column (5 mL, Cytiva). After sample application, the column was washed with 9 column volumes wash buffer (150 mM NaCl, 50 mM Tris, 25 mM imidazole, pH = 8.0) and eluted with 5 column volumes elution buffer (150 mM NaCl, 50 mM Tris, 500 mM imidazole, pH = 8.0). The elution was concentrated (\u0026lt; 2.5 mL) and applied to a HiLoad® 16/60 Superdex® 200 pg (Cytiva) size-exclusion column, which had been equilibrated with running buffer (150 mM NaCl, 50 mM Tris, pH = 8.0). Fractions containing the proteins, validated by SDS-PAGE, were collected, concentrated, frozen in liquid N\u003csub\u003e2\u003c/sub\u003e and stored at -80°C.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003ePurification of the ArnAB complex - Phosphorylation of ArnB\u003c/h2\u003e \u003cp\u003eFor the generation of the phosphorylated ArnAB complex a 1L cell pellet of untagged ArnC, which was expressed and harvested as described above, was added to the ArnA-ArnB coexpression cell pellets before lysis. In addition, after the heat step ATP (Sigma Aldrich) and MnCl\u003csub\u003e2\u003c/sub\u003e (2.5 mM final concentration each) were added to the supernatant before incubation in a water bath (55°C, 5–60 min). Purification of the phosphorylated complex was performed with Ni-NTA affinity chromatography and SEC as described above.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eCrystallization and structure determination of the ArnAB complex\u003c/h2\u003e \u003cp\u003eFreshly purified, equimolar protein of ArnA and ArnB (4.85 mg/mL) was filtered with a centrifuge reaction tube micro filter (Merck) prior to crystallization experiments. Initial crystal hits of the ArnAB complex grew in well F11 of the JCSG Core III Suite (NeXtal) but were unsuitable for structure determination. Accordingly, a fine screen hanging drop vapor diffusion experiment on 24-well plates by mixing 1 µL of crystallization condition with 1 µL protein solution was conducted. The final monoclinic crystals grew in the presence of 0.17 mol/L ammonium acetate, 0.085 mol/L sodium citrate pH 6.0, 25.5% (w/v) PEG 4000, 16.5% (v/v) glycerin after 5 months.\u003c/p\u003e \u003cp\u003eArnAB data set was collected at the Swiss Light Source at Paul Scherrer Institute (\u003cem\u003eSwitzerland\u003c/em\u003e). Structure determination of the ArnAB complex was done by molecular replacement with the vWA2 structure (PDB: 5A8J) from \u003cem\u003eSulfolobus acidocaldarius\u003c/em\u003e as search model. Afterwards, the structure was refined by multiple rounds of manual structure building with Coot \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e followed by phenix.refine \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e, which also led to electron density defining the ArnA ZnF-domain. Data collection and refinement statistics can be found in Table S2. Protein structure was deposited to the Protein Data Base and can be accessed via the code: 8SO5.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eSmall angle X-ray scattering (SAX)\u003c/h2\u003e \u003cp\u003eSAXS datasets were collected at the synchrotron Bio-SAXS beamline BM29 (ESRF) Grenoble, France \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. The wavelength \u003cem\u003eλ\u003c/em\u003e = 1.0 Å and the sample-to-detector distance of 2.43 m resulted in scattering vectors, q, ranging from 0.0025 Å\u003csup\u003e−1\u003c/sup\u003e to 0.50 Å\u003csup\u003e−1\u003c/sup\u003e. The scattering vector is defined as \u003cem\u003eq\u003c/em\u003e = 4π sin\u003cem\u003eθ\u003c/em\u003e/\u003cem\u003eλ\u003c/em\u003e, where 2θ is the scattering angle. All experiments were performed at 20°C and the data were initially processed by the ATSAS software package \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. SAXS data collection was performed in 150 mM NaCl, 50 mM Tris (pH 8) buffer. One-dimensional datasets were subtracted from the buffer-only spectrum then merged and analyzed in Primus \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. 1D-scattering of different concentrations were merged, where appropriate, given that the lower concentrations better represent low \u003cem\u003eq\u003c/em\u003e data points. The radius of gyration was calculated by ScÅtter \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. The \u003cem\u003eI\u003c/em\u003e\u003csub\u003e0\u003c/sub\u003e-values were calculated considering bovine serum albumin (BSA) as standard, where the \u003cem\u003eR\u003c/em\u003e\u003csub\u003eg\u003c/sub\u003e was 33.6 ± 0.4 Å and I\u003csub\u003e0\u003c/sub\u003e 93.13 ± 0.1. P(r) distance distribution functions were calculated and refined by the program ScÅtter. The distance \u003cem\u003er\u003c/em\u003e, where the \u003cem\u003eP\u003c/em\u003e(\u003cem\u003er\u003c/em\u003e) functions approach zero probability, identifies the maximal dimension (\u003cem\u003eD\u003c/em\u003e\u003csub\u003emax\u003c/sub\u003e) of the macromolecule. Using the refined data from ScÅtter in GNOM-format \u003cem\u003eab initio\u003c/em\u003e models for the corresponding SAXS-derived electron densities were calculated by averaging 20 rounds of DENSS \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. These electron densities were fitted with structures and finally visualized by ChimeraX. The SAXS data sets are deposited in SASBDB \u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e with accession codes SASDUW2.\u003c/p\u003e \u003cp\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eHydrogen/deuterium exchange mass spectrometry (HDX-MS).\u003c/span\u003e \u003c/p\u003e \u003cp\u003eInvestigated proteins (ArnA, ArnB, ArnB ~ P, ArnA/ArnB ~ P) were employed as 50 µM concentrated stocks solutions in 20 mM Tris-Cl pH 8.0, 150 mM NaCl. From these, HDX reactions were prepared by an autosampler (LEAP Technologies) as follows: 6.5 µL of protein stock solution were pre-dispensed in a 96-well plate, 58.5 µL of a buffer (20 mM Tris-Cl pH 8.0, 150 mM NaCl) prepared with 99.9% D\u003csub\u003e2\u003c/sub\u003eO added and incubated at 25°C for 10/30/100/1,000/10,000 s. The HDX reaction was stopped by transferring 55 µL of the reaction to 55 µL of quench solution (400 mM KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e/H\u003csub\u003e3\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e, 2 M guanidine-HCl (pH 2.2) pre-dispensed in another 96-well plate cooled down to 1°C. 95 µL of the quenched reaction were injected into an ACQUITY UPLC M-Class system with HDX Technology (Waters) operating at 0.5°C through a 50-µL injection loop. Non-deuterated samples were generated analogously with an H\u003csub\u003e2\u003c/sub\u003eO-based buffer.\u003c/p\u003e \u003cp\u003eSamples were washed out of the loop with water + 0.1% (v/v) formic acid (100 µL/min) and digested at 12°C in a cartridge (2 cm x 2 mm) filled with either bead-immobilized porcine pepsin or a 1:1 mixture of bead-immobilized protease type XVIII from \u003cem\u003eRhizopus\u003c/em\u003e sp. and protease type XIII from \u003cem\u003eAspergillus saitoi\u003c/em\u003e. The resulting peptides were collected on a trap column (2 mm x 2 cm; 0.5°C) filled with POROS 20 R2 reversed phase resin (ThermoFisher Scientific). After 3 min of digestion and trapping, the trap was placed in line with an ACQUITY UPLC BEH C18 1.7 µm 1.0 x 100 mm column (Waters) operating at 0.5°C and the peptides eluted with a gradient of water + 0.1% (v/v) formic acid (eluent A) and acetonitrile + 0.1% (v/v) formic acid (eluent B) at 30 µL/min, as follows: 0–7 min/95 − 65% A, 7–8 min/65 − 15% A, 8–10 min/15% A. The peptides were guided to a Synapt G2-Si mass spectrometer (Waters) and ionized by electrospray ionization (capillary temperature: 250°C; spray voltage: 3.0 kV). Mass spectra were acquired with MassLynX MS 4.1 (Waters) over 50 to 2,000 \u003cem\u003em/z\u003c/em\u003e in enhanced high-definition MS (HDMS\u003csup\u003eE\u003c/sup\u003e)\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e,\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e or high-definition MS (HDMS) mode for non-deuterated and deuterated samples, respectively. A short spray of [Glu1]-fibrinopeptide B standard (Waters) every 45 s was employed for lock-mass correction. During peptide separation, the protease column was washed three times with 80 µL of 0.5 M guanidine-HCl in 4% (v/v) acetonitrile, and blank runs (double-distilled H\u003csub\u003e2\u003c/sub\u003eO) were performed between each sample. All measurements were performed in triplicates for each protease column type (separate HDX reactions).\u003c/p\u003e \u003cp\u003ePeptides were identified ProteinLynx Global SERVER 3.0.1 (PLGS, Waters) and DynamX 3.0 (both Waters) from the non-deuterated samples acquired with HDMS\u003csup\u003eE\u003c/sup\u003e as described previously\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e employing low-energy, elevated-energy and intensity thresholds of 300, 100 and 1,000 counts, respectively and matched using a database containing the amino acid sequences of ArnA, ArnB, porcine pepsin, and their reversed sequences (peptide tolerance = automatic; fragment tolerance = automatic; min fragment ion matches per peptide = 1; min fragment ion matches per protein = 7; min peptide matches per protein = 3; maximum hits to return = 20; maximum protein mass = 250,000; primary digest reagent = non-specific; missed cleavages = 0; false discovery rate = 100). For quantification of deuterium incorporation with DynamX, peptides had to fulfil the following criteria: identification in two-thirds of the non-deuterated samples; minimum intensity of 30,000 counts; maximum length of 30 residues; minimum number of products of three and 0.1 products per residue; maximum mass error of 25 ppm; retention time tolerance of 0.5 minutes. Hereby, the datasets generated with porcine pepsin or after digestion with proteases type XIII and XVIII were pooled, and all spectra manually inspected. Residue-specific deuterium uptake of peptides was calculated with DynamX. If a residue was covered by a single peptide, the residue-specific deuterium uptake was equal to that of the whole peptide. In the case of overlapping peptides for a given residue, the residue-specific deuterium uptake was determined by the shortest peptide covering that residue. When multiple peptides were of the shortest length, the peptide with the residue closest to the C-terminus was utilized.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eProteomics data evaluation\u003c/h2\u003e \u003cp\u003eSample preparation and measuring of the \u003cem\u003eS. acidocaldarius\u003c/em\u003e proteomes was performed as previously described \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e in collaboration with the bioanalytics MarMass facility of the Philipps University of Marburg.\u003c/p\u003e \u003cp\u003eThe timsTOF data was analyzed utilizing MaxQuant 2.1.3. Sequencing information for \u003cem\u003eS. acidocaldarius\u003c/em\u003e DSM 639, comprising 2,222 entries, was retrieved from the UniProt database. For label-free quantification (LFQ) analysis, MaxQuant parameters were configured as follows: maximum peptide mass of 4,000 Da, allowance of up to three modifications per peptide, up to three missed cleavages, and the necessity of MS/MS for LFQ comparisons; all other parameters remained at default settings. Protein abundances from biological triplicates of the ΔArnA, ΔArnB and the wild type strain were filtered using a two-tailed student’s t-test (p \u0026lt; 0.05). The remaining proteins were visualized in protein heat maps, with further filtering based on a mean abundance difference of \u0026gt; 0.5 log\u003csub\u003e2\u003c/sub\u003e fold-change. Visualization was done using the pheatmap R package \u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e in addition to the gplots package \u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. The volcano plots were constructed by plotting the log\u003csub\u003e2\u003c/sub\u003e fold-change in protein abundance on the x-axis against the negative logarithm (base 10) of the p-values (calculated from the student’s t-test) on the ordinate. Plots were created using the ggplot2 \u003csup\u003e32\u003c/sup\u003e and ggrepel \u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e packages in R, providing a comprehensive visualization of the proteomic changes in the context of significance levels. Gene Ontology (GO) enrichment analysis was performed on the sets of significant proteins (p \u0026lt; 0.05) from each comparison using GOATOOLS library version 1.2.3 \u003csup\u003e34\u003c/sup\u003e. The significance of the GO enrichment analysis was determined by applying the Benjamini-Hochberg procedure with a false discovery rate (FDR) correction at a significance threshold of p = 0.05. The analysis was carried out with gene ontology terms specific to \u003cem\u003eS. acidocaldarius\u003c/em\u003e DSM639, obtained via the QuickGO \u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e annotation tool.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eMutagenesis of ArnB threonines\u003c/h2\u003e \u003cp\u003eFor point mutation of the different ArnB mutants 10–60 ng of starting vector was mixed with 150 ng of corresponding primers (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e), 1.5 µL DMSO, 1 µL dNTP mix (NEB), 0.5 µL of Phusion DNA-polymerase stock (~ 6 mg/mL) and adjusted to 50 µL with H\u003csub\u003e2\u003c/sub\u003eO. The PCR was performed for 18 cycles, primer suitable annealing temperatures (50–56°C) and an elongation temperature of 72°C. PCR was followed by a DpnI (2 µL, NEB) digest (37°C, 2h) and transformation into \u003cem\u003eE. coli\u003c/em\u003e DH5α cells. Afterwards plasmids were extracted from overnight cultures using the QIAprep Spin Miniprep Kit (Quiagen) and checked by Sanger sequencing.\u003c/p\u003e \u003c/div\u003e "},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor Contributions: L.K.:\u003c/strong\u003e Conceptualization, Formal analysis, Investigation, Methodology, Project administration, Validation, Visualization, Supervision of L.S., Writing \u0026ndash; original draft, and Writing \u0026ndash; review and editing; \u003cstrong\u003eW.S.:\u003c/strong\u003e Investigation of HDX experiments, Formal analysis of HDX data, Writing \u0026ndash; original draft, and Writing \u0026ndash; review and editing; \u003cstrong\u003eM.W.:\u003c/strong\u003e Formal analysis of MS data and Visualization; \u003cstrong\u003eF.B.:\u003c/strong\u003e Investigation, and Supervision of A.P.; \u003cstrong\u003eM.S.V.:\u003c/strong\u003e Investigation, and Project administration; \u003cstrong\u003eL.S.:\u0026nbsp;\u003c/strong\u003eInvestigation; \u003cstrong\u003eA.P.:\u003c/strong\u003e Investigation; \u003cstrong\u003eS.H.:\u003c/strong\u003e Investigation of SAXS experiments, and Formal analysis of SAXS data; \u003cstrong\u003eS.V.A.:\u003c/strong\u003e Funding acquisition, Project administration, Supervision, Writing \u0026ndash; original draft, and Writing \u0026ndash; review and editing; \u003cstrong\u003eL.O.E.:\u003c/strong\u003e Conceptualization, Data curation, Formal analysis, Funding acquisition, Project administration, Resources, Supervision, Writing \u0026ndash; original draft, and Writing \u0026ndash; review and editing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interest Statement:\u0026nbsp;\u003c/strong\u003eThe authors declare no competing interests.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eClassification:\u0026nbsp;\u003c/strong\u003eBiological Sciences / Biochemistry.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data can be found in the supplementary information text or excel files. Protein structures have been uploaded to the PDB.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank the beamline staff of the Swiss Light Source (SLS), PSI, Villigen, Switzerland, as well as the beamline staff of the ESRF, Grenoble, France, for their support. We also want to thank the staff of Marburg Mass spectrometry and crystallization facility (MarXtal) for technical support.\u0026nbsp;We acknowledge support by the German Research Council (DFG) through the core facility for HDX-MS (project numbers 260989694 and 324652314 to Gert Bange, Marburg). We want to\u0026nbsp;thank the Life? program of the Volkswagen Foundation for funding (Grant: Az-96727).\u003cbr\u003e\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eJarrell, K. F. \u0026amp; Albers, S. V. 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Together, they form either a loose or a tight complex, whose transition is directed by phosphorylation via the kinase ArnC. For a structure-based analysis of this transition we solved a cocrystal structure of the ArnA/ArnB complex revealing that the zinc finger domain of ArnA interacts with the β-sandwich and C-terminal domain of ArnB. HDX data corroborate the phosphorylation-dependent transition from loose to tight ArnAB complexes. This transition depends on a structural transformation of ArnB by sequential phosphorylation, exposing the interaction surface of the C-terminal domain of ArnB for the forkhead-associated domain of ArnA. Furthermore, we found a striking structural similarity between ArnB and the membrane-curving proteins of the COPII vesicle system, Sec23/Sec24. The common Sec23/Sec24 core motif can be found in all domains of life, where it can apparently adopt a multitude of different functions. Overall, this implies that Sec23/Sec24 orthologs with a function in vesicle formation arose in Lokiarchaeota from related, but not necessarily functionally linked relatives as found in TACK Archaea.\u003c/p\u003e","manuscriptTitle":"Sequential conformational transition of ArnB, an archaeal ortholog with Sec23/Sec24 core motif","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-03-11 08:39:03","doi":"10.21203/rs.3.rs-3955852/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"communications-biology","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"commsbio","sideBox":"Learn more about [Communications Biology](http://www.nature.com/commsbio/)","snPcode":"","submissionUrl":"","title":"Communications Biology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Communications Series","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"b96808ac-73d7-436d-91f6-220de80506a5","owner":[],"postedDate":"March 11th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2024-03-11T08:39:03+00:00","versionOfRecord":[],"versionCreatedAt":"2024-03-11 08:39:03","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3955852","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3955852","identity":"rs-3955852","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}