Nanobody-mediated modulation of long RSH enzymes Rel and RelA catalysis by restriction of their conformational landscape

Nanobody-mediated modulation of long RSH enzymes Rel and RelA catalysis by restriction of their conformational landscape | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var 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Talavera-Perez , View ORCID Profile Dannele Echemendia-Blanco , Sarah Peeters , Brahim El Khalfaoui Oulali , View ORCID Profile Hedvig Tamman , View ORCID Profile Tatsuaki Kurata , View ORCID Profile Mohammad Roghanian , View ORCID Profile Chloé Martens , View ORCID Profile Els Pardon , View ORCID Profile Jan Steyaert , View ORCID Profile Vasili Hauryliuk , View ORCID Profile Abel Garcia-Pino doi: https://doi.org/10.1101/2025.08.07.669093 Katleen Van Nerom 1 Cellular and Molecular Microbiology, Faculté des Sciences, Université libre de Bruxelles (ULB) , 1050 Brussels, Belgium Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Katleen Van Nerom Andres Ainelo 1 Cellular and Molecular Microbiology, Faculté des Sciences, Université libre de Bruxelles (ULB) , 1050 Brussels, Belgium 2 Department of Genetics, Institute of Molecular and Cell Biology, University of Tartu , Tartu, Estonia Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Andres Ainelo Kyo Coppieters ’t Wallant 3 Biochemistry and Structural Biology, Université libre de Bruxelles (ULB) , 1050 Bruxelles, Belgium Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Kyo Coppieters ’t Wallant Ariel Talavera-Perez 1 Cellular and Molecular Microbiology, Faculté des Sciences, Université libre de Bruxelles (ULB) , 1050 Brussels, Belgium Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Ariel Talavera-Perez Dannele Echemendia-Blanco 1 Cellular and Molecular Microbiology, Faculté des Sciences, Université libre de Bruxelles (ULB) , 1050 Brussels, Belgium Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Dannele Echemendia-Blanco Sarah Peeters 1 Cellular and Molecular Microbiology, Faculté des Sciences, Université libre de Bruxelles (ULB) , 1050 Brussels, Belgium Find this author on Google Scholar Find this author on PubMed Search for this author on this site Brahim El Khalfaoui Oulali 1 Cellular and Molecular Microbiology, Faculté des Sciences, Université libre de Bruxelles (ULB) , 1050 Brussels, Belgium Find this author on Google Scholar Find this author on PubMed Search for this author on this site Hedvig Tamman 1 Cellular and Molecular Microbiology, Faculté des Sciences, Université libre de Bruxelles (ULB) , 1050 Brussels, Belgium 2 Department of Genetics, Institute of Molecular and Cell Biology, University of Tartu , Tartu, Estonia Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Hedvig Tamman Tatsuaki Kurata 4 Department of Experimental Medical Science, Lund University , Lund, Sweden 5 RNA Systems Biochemistry Laboratory, Pioneering Research Institute , RIKEN, Wako, Saitama 351-0198, Japan Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Tatsuaki Kurata Mohammad Roghanian 6 Department of clinical microbiology, Rigshospitalet , Copenhagen, Denmark Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Mohammad Roghanian Chloé Martens 3 Biochemistry and Structural Biology, Université libre de Bruxelles (ULB) , 1050 Bruxelles, Belgium Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Chloé Martens Els Pardon 7 VIB-VUB Center for Structural Biology , VIB, Pleinlaan 2, B-1050 Brussels, Belgium 8 Structural Biology Brussels, Vrije Universiteit Brussel , Pleinlaan 2, B-1050 Brussels, Belgium Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Els Pardon Jan Steyaert 7 VIB-VUB Center for Structural Biology , VIB, Pleinlaan 2, B-1050 Brussels, Belgium 8 Structural Biology Brussels, Vrije Universiteit Brussel , Pleinlaan 2, B-1050 Brussels, Belgium Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Jan Steyaert Vasili Hauryliuk 4 Department of Experimental Medical Science, Lund University , Lund, Sweden 9 NanoLund and Science for Life Laboratory, Lund University , Lund, Sweden 10 University of Tartu, Institute of Technology , Tartu, Estonia Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Vasili Hauryliuk Abel Garcia-Pino 1 Cellular and Molecular Microbiology, Faculté des Sciences, Université libre de Bruxelles (ULB) , 1050 Brussels, Belgium 11 WELRI: WEL Research Institute , avenue Pasteur, 6, 1300 Wavre, Belgique Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Abel Garcia-Pino For correspondence: abel.garcia.pino{at}ulb.be Abstract Full Text Info/History Metrics Preview PDF Abstract Long RSH enzymes, Rel and RelA, are the master regulators of (p)ppGpp alarmone levels in bacteria. Their catalytic activity is governed by transitions between a compact, hydrolysis-competent (HD ON ) state and an elongated, synthesis-competent (SYNTH ON ) state. The equilibrium between these states is modulated by factors such as “starved” ribosomes and regulatory proteins DarB, EIIA NTR , ACP and YtfK. Here, we identify and characterize camelid nanobodies that act as selective allosteric modulators by trapping Rel/RelA enzymes in distinct conformational states. Nanobodies that lock the TGS domain of RelA and prevent its activation by deacylated tRNA on starved ribosomes, strongly inhibit (p)ppGpp synthesis and suppress the virulence of E. coli in an animal model. Nb898 stabilizes Rel in the open SYNTH ON state, enhancing synthesis activity while suppressing hydrolysis. Conversely, Nb585 traps Rel in a HD ON conformation, strongly inhibiting alarmone synthesis while promoting (p)ppGpp hydrolysis. Structural and biochemical analyses reveal that nanobodies, like natural allosteric regulators, act by restricting the RSH enzyme’s conformational landscape. These findings establish nanobodies as powerful tools for dissecting RSH function and provide potential leads for developing protein-based RSH modulators. Introduction Nucleotides guanosine-3’,5’-tetraphosphate and guanosine-3’5’-pentaphosphate – collectively referred to as (p)ppGpp alarmones – play a key role in bacterial stress sensing and adaptation 1 , 2 . The alarmones control different aspects of physiology including the growth rate, metabolism, transcription and translation and are effective at different cellular concentrations 3 – 8 . As master regulators of bacteria physiology, the alarmones are also implicated in control of bacterial virulence, biofilm formation and tolerance to antimicrobials 9 – 13 . The balance between synthesis and hydrolysis of (p)ppGpp is tightly controlled. The primary enzymes controlling (p)ppGpp homeostasis are “long” Rel/RelA/SpoT homolog enzymes, collectively known as long RSHs 14 , 15 . These enzymes are near-universal in bacteria, present in genomes of the majority of species, with an exception of Plantomycetes, Verrucomicrobia and Chlamydiales, the PVC superphylum 14 . Long RSHs contain two catalytic or pseudo-catalytic domains in their N-terminal half (NTD): a hydrolysis domain (HD; or pseudo-HD if inactive) and a synthesis domain (SYNTH; or pseudo-SYNTH if inactive). Their enzymatic activity is regulated by four C-terminal regulatory domains (CTD) 16 – 20 . In the case of Rel and RelA, the primary signal that stimulates (p)ppGpp production is amino acid starvation, which is sensed by recognizing the deacylated (uncharged) tRNA in the ribosomal A site 21 , 22 . In addition to this in cis intra-protein regulation, the SYNTH activity of Rel/RelA is also stimulated in trans by the alarmones themselves, with pppGpp being a more potent activator 17 , 23 , 24 . A complex allosteric network controls and finetunes the enzymatic output of long RSH enzymes. In the case of bifunctional (capable of both degrading and synthesizing the alarmones) RSH enzyme Rel the opposing activities of both catalytic domains are controlled by the relative concentrations of substrates, with the ligand binding to either of the active sites switching off the activity of the other 19 . Importantly, the allosteric effect of the CTD supersedes the crosstalk between catalytic NTD domains 17 , 25 . In the HD ON state, the enzyme is in a closed conformation, in which the CTD activates hydrolysis by stabilizing the HD domain and promoting the organization of the active site while precluding the activation of the SYNTH domain 18 . Upon amino acid starvation, binding of the enzyme to “starved” (i.e. containing a deacylated tRNA in the A site) ribosome in a fully extended elongated state abolishes the allosteric effect of the CTD on hydrolysis and exposes the SYNTH domain 26 , 27 . However, the complete activation of the SYNTH domain also requires the binding of alarmones to a dedicated allosteric site that, acting in concert with the A-site-bound uncharged tRNA, stabilizes an elongated ribosome-bound state of the enzyme, triggering the processive synthesis of (p)ppGpp 17 , 25 . By contrast, active hydrolysis is contingent on a compact τ-state of long RSH enzymes Rel and SpoT, in which the SYNTH active site is occluded and the HD catalytic residues and metal co-factor are stabilized and primed for (p)ppGpp binding and hydrolysis 18 , 19 . In addition to being controlled by starved ribosomal complexes, NTD and CTD regions are the target of diverse regulatory proteins the modulate the activity of RSHs 28 – 34 . While EIIA NTR , ACP and YtfK were reported to inhibit the hydrolase function of Rel and SpoT 31 , 34 , 35 and the synthetase activity of RelA 36 , DarB activates the synthetase activity of Rel in a ribosome-independent manner 37 . This suggests that long RSH enzymes possess a network of allosteric “hotspots” that modulate the enzymes’ catalytic activities. Such hotspots would allow long RSH enzymes to sense the bacterial metabolic state while simultaneously providing the checkpoints that are crucial to control overproduction of (p)ppGpp 17 , 18 . Camelid nanobodies are well stablished structural biology tools used to study the conformational landscape of proteins 38 – 40 . Here, we leveraged nanobodies to investigate the conformational dynamics of Rel and RelA, identifying allosteric hotspots that can be targeted to modulate their catalytic activity. Our structural and biochemical analyses reveal that by locking Rel/RelA enzymes in specific conformational states, we can precisely manipulate its activity. We have identified and characterized high affinity nanobodies that can mimic the effect of 70S ribosomes on Rel’s synthetase activity by binding to an allosteric site on the SYNTH domain. In contrast, nanobodies that interact with the catalytic domains promote hydrolysis and strongly inhibit synthesis by stabilizing the enzyme’s τ-state. These findings highlight the potential of targeting allosteric sites outside the active centers of RSH enzymes, paving the way for novel antimicrobial strategies against stringent response regulators. Results Generation of high affinity nanobodies against RSH enzymes While the structures of the extended conformation of the ribosome-bound state of the (p)ppGpp synthetases Rel and RelA are well characterized 26 , 27 , their off-ribosome state remains refringent to structural biology. Only the structure of the monofunctional hydrolase SpoT from Acinetobacter baumannii was recently determined 18 , which a revealed a compact τ-shaped architecture incompatible with alarmone synthesis. To generate the nanobodies we could use to explore the conformational landscape of the long RSH synthetases off the ribosome, we immunized different llamas with Escherichia coli RelA (RelA Ec ) and Rel from Chlorobaculum tepidum (Rel Ct ), and obtained camelid nanobodies after phage display selection using established protocols 41 . After two rounds of selection against RelA Ec and Rel Ct – either in unbound state or in the presence of GDP or APCPP – we have successfully isolated a set of different candidate binders that were classified according to the sequences of the third complementarity determining region (CDR3, see Supplementary Fig. 1a ). RelA Ec -targeting nanobodies inhibit (p)ppGpp synthesis by E. coli RelA in vivo (p)ppGpp alarmones are essential for maintaining the cellular homeostasis, specifically for controlling the amino acid synthesis 15 , 42 . This key role of (p)ppGpp is leveraged in so-called SMG functional test for RelA SYNTH activity: only E. coli expressing SYNTH-competent RSH can grow in the SMG defined medium 43 . Therefore, to characterize the anti-RelA Ec nanobodies, we have first expressed them in E. coli and monitored the effect on bacterial growth either under permissive conditions (M63 medium 44 , under these conditions inhibition of RelA SYNTH activity does not affect the growth) or under non-permissive conditions (SMG medium, under these conditions inhibition of RelA SYNTH activity abrogated growth). None of nanobodies were toxic when expressed in bacteria growing on M63 media ( Fig. 1a ). Conversely, IPTG-inducible expression of Nb94 fully abrogated the E. coli DJ624 growth on SMG medium but not in M63 ( Fig. 1a ). Nb96 displayed a mild SMG-specific inhibitory effect and Nb120, a control nanobody selected against a different target, did not inhibit growth on SMG. Next, we assessed the interactions between nanobodies and full-length E. coli RelA (RelA Ec ) in vitro using Isothermal Titration Calorimetry (ITC). Nb94 and Nb96 bound RelA Ec with affinities of 13 nM and 157 nM, respectively, and no interaction was detectable for Nb120 ( Fig. 1b-d and Supplementary Table S1 ). Thus, the high affinity of Nb94 and Nb96 to RelA were consistent with the SMG assays that showed these are potent inhibitors of RelA’s SYNTH activity in vivo . Download figure Open in new tab Fig. 1. Nanobodies against RelA prevent growth of E. coli under amino acid starvation conditions and curtail virulence. (a) SMG RelA functionality test of cells transformed with vectors expressing Nb94, Nb96 and the control Nb120 used as negative control. ITC titrations of Nb94 (b) , Nb96 (c) , and Nb120 (d) into RelA. (e) Virulence assays in the G. mellonella infection model demonstrate nanobodies that inhibit RelA affect strongly the virulence of E. coli . (f) AlphaFold predicted structural model of the complex of RelA; coloured from N to C-terminus: NTDs pseudo-HD (light blue), SYNTH (in gold) and Core (in red), and CTDs, TGS (teal), HEL (pink), ZFD (dark navy blue) and RRM (purple) and Nb94 (in kaki). (g) Details of the interaction of Nb94 (kaki) and Nb96 (orange) with RelA’s TGS domain (coloured in teal and turquoise for each respective complex). The orientation of the nanobodies in both complexes underline the importance of H432 (labeled in the figure) to the binding interface. Prediction scores are shown in the figure. Nb94 and Nb96 curtail virulence of E. coli (p)ppGpp-mediated signaling is crucial in antibiotic tolerance and virulence of E. coli . We reason that our RelA-modulating nanobodies Nb94 and Nb96 could have an impact on the virulence of E. coli mimicking the effects of genetic knockouts catalytically impaired variants of RelA. We used the wax moth Galleria mellonella larvae infection model to assess this effect on the E. coli clinical isolate Ec156 45 . In the presence of the control Nb120 (produced by the Ec156_nb120 strain), which was not selected against RelA, only 30% of the larvae survived the infection after 36h. By contrast when the Galleria larvae were challenged with the Ec156_nb94 and Ec156_nb96 (Ec156 strains 45 transformed with plasmids expressing the Nb94 and Nb96 nanobodies) survival increased to 50% and 70% respectively ( Fig. 1e ). Notably, these strains displayed no growth defects when grown on M63 plates. Our infection assays demonstrate that while E. coli can tolerate the loss of activity of RelA when grown in nonstressed laboratory conditions without a dramatic fitness defect, a fully functional (p)ppGpp synthetase is crucial for a successful infection. Nb94 and Nb96 block the TGS of RelA Ec To gain a functional insight into the nanobody’s mode of action, we have used AlphaFold 46 to model the structure of the RelA Ec :Nb94 and RelA Ec :Nb96 complexes. High confidence models predict that Nb94 and Nb96 recognize RelA Ec via the TGS through a ≈990 Å 2 and ≈850 Å 2 interface respectively ( Fig. 1f-g ). The complementarity-determining regions 2 and 3 of Nb94 and Nb96 provide the majority of the contacts (with CDR1 not involved in binding) by interfacing with the Threonyl-tRNA Synthetase, GTPase and SpoT (TGS) domain of RelA through the central α-helical region and the C-terminal β-strand, as well as via additional contacts with the α6-α7 motif of the pseudo-HD domain and the Core domain. Interestingly, the complex interface is dominated by the H432 residue located in an antiparallel α-helical motif of the TGS that makes extensive contacts with CDR2 and CDR3 and β-strands β3 and β4 Nb94 ( Supplementary Fig. 1a ). In the case of Nb96, the AlphaFold-predicted mode of TGS recognition differs. Nb96 engages the opposite face of the antiparallel α-helical motif within the TGS domain through all three CDRs, and additionally establishes extensive contacts via β-strands β3, β4, and β5. Notably, both nanobodies interact directly with residue H432, although in the case of Nb96, this interaction occurs specifically through CDR2. ( Fig. 1g and Supplementary Fig. 1b ). These differences in recognition reflect substitutions within the complementarity-determining regions, particularly in CDR2 ( Supplementary Fig. 1c ). Nevertheless, as both Nb94 and Nb96 nanobodies target the TGS domain of RelA, they are likely to disrupt the allosteric crosstalk between the catalytic and regulatory regions. Furthermore, their binding provides a structural basis for the potent inhibition of synthesis observed with Nb94 and Nb96, as they are predicted to tether the TGS domain to the pseudo-HD and Core domains, thereby likely preventing the enzyme from adopting its active, open conformation 26 , 27 , while also blocking the recognition of uncharged tRNAs on the A site by the TGS domain which is mediated by H432 47 . We used ITC to monitor the interaction of Nb94 and Nb96 with an H432E-substituted version of RelA Ec (RelA Ec H432E ), a well characterized variant that abrogates RelA’s functionality by abolishing RelA activation by deacylated tRNA 17 , 47 . In the case of the interaction of RelA Ec H432E with Nb94 and Nb96, the introduction of a negative charge to the interface is predicted to disrupt binding. Indeed, the lack of binding of either nanobodies to RelA H432E ( Supplementary Fig. 1d-e ) supports the proposed mode of recognition and inhibition of ppGpp synthesis through sequestration of the TGS domain. Nanobodies modulate Rel Ct catalytic activities While the monofunctional SYNTH-only RelA can only explore the resting and extended states observed in long RSH ppGpp synthetases, bifunctional SYNTH- and HD-competent Rel enzymes have a much broader conformational landscape 18 . Being capable of both, hydrolysis and synthesis of (p)ppGpp, these proteins readily explore different conformational states 18 . These states involve a compact HD ON -compatible τ-state, in which the NTD are close together to with synthetase domain completely occluded. The enzyme also explores a relaxed state (less structured in comparison with the τ-state) in which both catalytic domains are partially open and marginally active; and an elongated, ribosome-bound state, SYNTH ON -compatible, in which the NTD is fully open and the hydrolase active site is disordered and inactive 18 , 19 . Therefore, discovering and engineering specific conformational modulators of Rel is in principle more complex. We turned to the bifunctional (p)ppGpp synthetase/hydrolase Rel from the model thermophile C. tepidum (Rel Ct ) to test whether nanobodies could selectively restrict the allosteric landscape of long RSH factors to trap the enzyme in a specific state that would define its catalytic output. Using the same strategy to identify nanobodies targeting RelA Ec , we selected two families of nanobodies – Nb898 and Nb585 – that strongly bind Rel Ct ( Supplementary Fig. 2 ). ITC showed that full-length Rel Ct interacts with Nb898 and Nb585 with K d values of 186 nM and 21 nM, respectively ( Fig. 2a-b and Supplementary Table S1 ). In both cases this interaction is confined to the N-terminal catalytic region (NTD) of the enzyme since C-terminal truncations did not affect binding ( Supplementary Table S1 ). In the absence of Rel SYNTH stimulators, pppGpp or 70S E. coli ribosomes, Rel Ct is a poor catalyst of ppGpp synthesis ( Fig. 2c ). Addition of E. coli 70S ribosomes and pppGpp increases the SYNTH activity ≈8 times. In the presence of Nb898, the turnover of Rel Ct increased ≈11-fold, on par with the enhancing effects of the 70S and pppGpp combined. This effect is accompanied by a 1.6-fold decrease in hydrolase activity ( Fig. 2d ) that results in the accumulation of ppGpp. By contrast, Nb585 turns SYNTH OFF , strongly inhibiting (p)ppGpp synthesis ( Fig. 2c ) while increasing the hydrolase activity of Rel Ct by two-fold ( Fig. 2d ). These results are consistent with the nanobodies modulating the antagonistic allosteric coupling between the two catalytic domains of Rel Ct to control the enzymatic output. Download figure Open in new tab Fig. 2. Nanobodies targeting Rel can modulate the activity of the enzyme by restricting their allosteric landscape. ITC titrations of Nb898 (a) , Nb585 (b) , into Rel Ct . (c) SYNTH activity of Rel Ct unbound (empty bar); in the presence of Nb898 (red bars); Nb585 (blue bars); pppGpp (grey) and 70S ribosomes (black). (d) HD activity of Rel Ct unbound, in the presence of Nb898 (red bars), Nb585 (blue bars). The contrasting features of the action of the nanobodies in (c) and (d) suggest they highjack the enzyme’s conformational landscape to favour one activity in detriment of the other. (e) Deuterium uptake profiles for representative peptides the allosteric hotspot region involving residues 118-127 of α6α7 (e) and residues 209-221 of α11 (f) . Nb898 increases Rel NTD dynamics We monitored the structural effects of Nb898 binding using Hydrogen Deuterium eXchange coupled with Mass Spectrometry (HDX-MS) by comparing Rel NTD (a more stable and soluble fragment that contains the Nb binding site) in its substrate-free unliganded state, and in the Rel NTD :Nb898 complex ( Supplementary Fig. 3a ). The overall effect of Nb898 binding was an increased deuterium uptake (ΔHDX) involving both catalytic regions that radiates from the central region of the enzyme ( Fig. 2e-f and Supplementary Fig. 3a ). The highest uptake increase localizes to the allosteric hub involving the α6α7 active site motif (residues 101 - 135) and α11 (residues 201 - 236) that mediate the coupling between SYNTH and HD domains 17 , 19 . The higher degree of uptake observed in this crucial allosteric region ( Fig. 2e-f ) suggests that the binding of Nb898 triggers a more dynamic state of the enzyme in which the SYNTH active site is more exposed and primed for substrate binding and catalysis. This aligns closely with prior structural evidence showing that α11 unwinds in Rel and RelA upon activation of the SYNTH domain 17 , 19 . The HDX-MS data also detected a prominent decrease in deuterium exchange in the region that contains α-helix α13 (residues E279 to Y290), which is indicative of the direct association of Nb898 with Rel NTD and showed a slight additional deuterium protection signal in the hydrolase active site (residues 65-78, Supplementary Fig. 3a ). A more moderate protection was observed towards the C-terminal in the region. This involved Rel’s G-loop (residues 304 - 316, Supplementary Fig. 3a ), suggesting a possible allosteric stabilization of the residues involved in GDP/GTP binding that could stimulate catalysis. To confirm the binding interface, we probed the epitope define by α13 using point substitutions in Rel Ct (K280A, D283A, F285A, A286K, Y290A) to monitor the interaction with Nb898 by ITC. In all cases we detected an increase in the binding K d of the Rel Ct variants compared to the WT Rel Ct confirming the binding epitope ( Fig. 3a-d and Supplementary Table S1 ). Collectively these results indicated that the direct interaction of Nb898 with the SYNTH domain triggered the closing or rigidification of HD and consequent inactivation of the hydrolase function and activated the SYNTH function by allosterically increasing backbone dynamics. Download figure Open in new tab Fig. 3. Nb898 binds to the SYNTH domain of Rel Ct contacting α13 and the G-loop. ITC titrations of Nb898 into Rel Ct K280A (a) , Rel Ct D280A (b) , Rel Ct A286K (c) , and Rel Ct Y290A (d) . into Rel Ct validate the binding interface. (e) Crystal structure of the Rel Ct NTD -Nb898 complex. Rel’s HD and SYNTH domains are coloured as in Fig. 1f , and Nb898 in red. CDR2 and CDR3 are labeled. (f) Details of Rel Ct NTD -Nb898 binding interface highlighting key structural elements. (g) Superposition of the catalytic domains of Rel Ct onto the Rel Tt -AMP-ppGpp complex (PDBID 6S2U, in green) and the Rel Bs -DarB complex (PDBID 8ACU, in purple). The superposition confirms Nb898 selects the open, SYNTH ON conformation of the enzyme. The AMP and ppGpp nucleotides observed in the post-catalytic state of Rel Tt are are highlighted with light and dark grey arrows respectively. Nb898 stabilizes the SYNTH ON active state of Rel Ct NTD In order to understand the effect of Nb898 on catalysis, we determined the structure of the Nb898:Rel Ct NTD complex ( Fig. 3e ). Inspection of the complex confirms the binding interface predicted by the HDX-MS: indeed, Nb898 engages Rel Ct NTD via the SYNTH domain. The interactions from the nanobody side occur mainly through the core β-sheet of the Ig-domain, CDR2 which forms a flat hairpin structure with a pronounced bulge in the middle stacking α13, plus additional contribution from CDR3 ( Fig. 3e ). This binding interface is in perfect agreement with the HDX and mutagenesis results ( Fig. 2e and 3a-d ). The stacking interface is largely hydrophobic, composed by the side chains of residues I281, F285, A286, Y290 and T292 from the Rel Ct side and Y37, A47, V50, A52, D55 and T59 from Nb898. In addition, T59 forms a strong hydrogen bond with D283 tethering CDR2 to α13 and the backbone of T58 hydrogen-bonds K280 ( Fig. 3f ). In the conformation of Rel Ct NTD induced by Nb898, the HD and SYNTH domains are distanced 47 Å ( Fig. 3e ). The active site of the HD domain is largely misaligned with the ED catalytic motif away from the Mn 2+ ion and the pocket that coordinates the 3’-pyrophosphate of (p)ppGpp during hydrolysis. By contrast, the SYNTH domain active site and catalytic residues are exposed to the solvent. This is accompanied with the fracturing of α11 (into α11’ and α11) and the displacement of α12, all signature structural elements of the activation of catalysis by the SYNTH domain ( Fig. 3e ). Noteworthy, this mode of recognition via α13 mimics that of Thermus thermophilus Rel (Rel Tt ) bound to the reaction products ppGpp and AMP 19 ( Fig. 3g ) as well as of B. subtilis Rel (Rel Bs ) bound DarB in absence of cAMP 48 ( Fig. 3g and Supplementary Fig. 3b-c ); the latter also mediates the activation of ppGpp synthesis and inhibition of hydrolysis 48 ( Fig. 3g ). Catalysis by long RSH enzymes is directly linked to their conformational state 18 , 27 . Binding of GDP and ATP trigger the opening of the NTD region exposing the SYNTH active site while binding of ppGpp exposes the HD domain and inactivates SYNTH 19 . Using GDP and APCPP to induce the active SYNTH state of Rel Ct NTD , we determined the structure of the substrate-liganded complex at 2.65 Å. The structure shows the binding of both nucleotides, indeed, promotes the open conformation ( Fig. 4a and Supplementary Fig. 3d-e ). The guanosine base of GDP is stacked by the G-loop Y314 and the 5’-diphosphates stabilized by residues K255, K261, and K303. APCPP is held deep into the active site ( Fig. 4b ). The adenine base is strongly coordinated by R245 and R273 whereas the triphosphate moiety is locked in position for catalysis by R245, K247, S251 and K255, bringing the β-phosphate close to the 3’-OH of GDP ( Fig. 4b ). This open state bound to the substrates matches strongly the conformation of Rel Ct NTD observed in the complex with Nb898 ( Fig. 4a ). Collectively these results indicate that Nb898 triggers the SYNTH ON conformation of Rel NTD even in absence of substrates. Download figure Open in new tab Fig. 4. Nb898 binds to the SYNTH domain of Rel Ct and allosterically enhances the affinity of the enzyme for GDP. (a) Superposition of the structure of the Rel Ct NTD -Nb898 complex (coloured as in Fig. 1f ) on the structure of Rel Ct NTD (in pink) in complex with APCPP (in purple) and GDP (in green). Crucial structural elements involved in the ON/OFF switch of the enzyme such as α11, α13 are labeled in the figure. The superposition confirms Nb898 triggers an active state of Rel Ct NTD compatible with nucleotides binding. (b) Details of Rel Ct NTD -APCPP-GDP complex interface highlighting key active site residues involved in substrate binding and catalysis. (c) ITC titration of APCPP into Rel Ct NTD ; (d) APCPP into Rel Ct NTD -Nb898 complex; (e) GDP into Rel Ct NTD ; (f) GDP into Rel Ct NTD -APCPP complex; (g) GDP into Rel Ct NTD -Nb898 complex. (h) Cartoon representation of a model of full-length Rel Ct in the hydrolase-compatible τ state. The SYNTH (in gold) and RRM (purple) domains are labeled in the figure. (i) Surface representation of an idealized (unrealistic) model of full-length Rel Ct NTD in complex with Nb898 (in red). The model confirms that the Rel Ct NTD -Nb898 complex is incompatible with the hydrolase-active τ state. Nb898 stimulates SYNTH activity Rel NTD by promoting nucleotide binding RSH enzymes typically display a preferential order for the incorporation of substrates to the SYNTH active site 19 , 48 , 49 . We used ITC to probe the incorporation of nucleotides to Rel NTD . Rel NTD binds the APCPP nonhydrolyzable ATP analogue with an affinity of 37.9 μM, which is enhanced by 1.3-fold in the presence of Nb898 ( Fig. 4c-d ). By contrast, in the absence of APCPP or Nb898, we were not able to detect binding of GDP to Rel NTD ( Fig. 4e ). In the presence of saturating APCPP concentration (5-fold above the K d ) the enzyme binds the GDP substrate with a K d of 69.8 μM, and Nb898 has a 10-fold stimulatory effect on the interaction ( K d = 7.0 μM) ( Fig. 4f-g ). These results indicate that Rel NTD binds its substrates in a sequential and ordered manner, initiated by ATP and followed by GDP or GTP. In addition, they suggest that the stabilizing presence of Nb898 enhances the affinity of the enzyme for both nucleotides, which likely underlines the SYNTH-stimulatory effect of the nanobody. Nb898 modulates Rel Ct τ-state precluding CTD autoinhibition and HD activation To further investigate the mechanism of allosteric activation of Rel Ct by Nb898, we modeled the structure of unliganded full-length Rel Ct based on the structure of the highly compact τ-state of A. baumannii SpoT (SpoT Ab ) which represents a CTD-autoinhibited long RSH primed for hydrolysis ( Fig. 4h ). Indeed, the integrity of the association of the CTD of long RSHs with its HD and SYNTH domains is crucial for the stability of the SYNTH-inhibited τ-state (SYNTH OFF ) of SpoT and Rel as evidenced by a decrease in their hydrolase activity upon destabilizing mutations or domain deletions 18 , 25 , 50 . This contrast with the SYNTH activity: the mechanism of activation of Rel, and the role of the CTD in preventing this activity, are only relevant in the context of starved ribosomes with the CTD autoinhibition being only a part of a process that also involves the activation and stabilization of the fully active enzyme by A-site tRNAs and pppGpp. While the CTD itself is intricately involved in ribosome binding, in the absence of ribosomes, CTD truncations do exhibit an increase in ppGpp synthesis 20 , 25 . In addition, as observed for the Rel activator DarB 48 , a ribosome-independent stabilization of an open, elongated state of Rel could have an impact in the SYNTH activity of the enzyme 48 . When we compared the structure of the Rel Ct -Nb898 complex with the model of SYNTH OFF Rel Ct it becomes apparent that both states are incompatible. The strong binding of Nb898 would clearly interfere with the RRM:SYNTH association which would drive the conformational equilibrium of the enzyme toward a relaxed non-autoinhibited state more compatible with SYNTH activity ( Fig. 4i ). Collectively these results suggest that Nb898 hijacked a naturally existing allosteric pathway of activation of long RSH factors. In this way, Nb898 restricted the conformational landscape of the enzyme to the SYNTH open state, allosterically priming catalytic residues and enhancing substrate recognition. This resulted in an increased ppGpp synthesis. Nb585 locks Rel Ct and primes the HD domain while switching off the SYNTH domain As with the SYNTH-activating Nb898, we used HDX-MS to characterize the interaction of the HD-activating Nb585 with Rel Ct . In the presence of Nb585, we observed deuterium protection signal clustering in the central region of the enzyme that comprises the C-terminal part of the HD domain that mediates the coupling between SYNTH and HD domains and α11. These structural elements are all solvent exposed in the open pre-catalytic state of Rel Ct when bound to GDP and APCPP and in the complex with Nb898. However, these regions coalesce together around the central axis of the enzyme in a closed state and become protected from deuterium exchange ( Fig. 5a-b ). By contrast in the HD domain, the active site shows moderate increase in deuterium uptake consistent. This becomes apparent in the regions containing K47-R48 involved in guanosine coordination and H57, H82, D83 involved in Mn 2+ coordination ( Fig. 5a and c-d ). Collectively these results are consistent with the exposure of catalytic residues upon binding to Nb585 and the stabilization of a HD ON state. Download figure Open in new tab Fig. 5. Nb585 contacts both HD and SYNTH domains of Rel Ct stabilising a closed conformation that is compatible with ppGpp hydrolysis. (a) Heatmaps representing the HDX of Rel Ct NTD (top), Rel Ct NTD -Nb585 complex (center) and ΔHDX (bottom). Residues involved in the binding interface are outlined by a dashed light blue line. (b) Deuterium uptake profile for a representative peptide the allosteric hotspot region involving residues 209-221 of α11 of Rel Ct NTD -Nb585 complex in blue vs Rel Ct NTD -Nb898 in red. Deuterium uptake profile for a representative peptide of the α6/α7 loop (c) and the active site fragment of α4 (d) . (e) Surface representation of Rel Ct NTD (in light grey) bound to Nb585 (in blue) as predicted by AlphaFold. Rel Ct NTD residues involved in the binding interface are coloured in dark blue. (f) Cartoon representation of the model of Rel Ct NTD -Nb585 complex with Rel Ct NTD HD and SYNTH domains coloured as in Fig. 1f and Nb585 in blue. The Mn2+ ion, a proxy for the (p)ppGpp binding site in the HD domain is highlighted in the figure. (g) ITC titration of Nb585 R215E substituted version (Nb585 R215E ) into Rel Ct NTD . This position is predicted by AlphaFold and HDX-MS to be involved in the complex interface. The increase in K D triggered by the R215E substitution supports the predicted mode of action of Nb585. RFU, relative fractional uptake. The HDX-MS data are in good agreement with the AlphaFold prediction of the structure of the complex that places Nb585 between HD and SYNTH domains. In the predicted complex Nb585 interacts with the SYNTH domain via the region involved in ATP, reminiscent of the way toxic Small Alarmone Synthetases (toxSAS) are inhibited by their antitoxins 51 , which is consistent with the strong inhibition of synthesis displayed by Nb585. This binding mode tightly tethers the HD and SYNTH domains ( Fig. 5e-f ) stabilizing a closed state that resembles that of Rel Tt bound to ppGpp 19 ( r.m.s.d of 0.9 Å, Supplementary Fig. 4a ). According to the model and consistent with the HDX-MS data, Nb585 makes extensive contacts with the C-terminal α-helix of the HD domain and α-helix α11 of SYNTH. In particular, R215 of Rel Ct is predicted to form a salt bridge with D31 of Nb585 ( Supplementary Fig. 4b-c ). This region contains the kink implicated in the closed/open switch of the NTD and is strongly protected from deuterium exchange upon Nb585 binding ( Fig. 5a-b ). To challenge this, we introduced the R215E substitution in Rel Ct and observed a 28-fold drop in affinity of Nb585 for Rel Ct R215E ( Fig. 5g ). This is consistent with our model of the complex ( Fig. 5e ) and the HDX-MS and biochemical data ( Fig. 2c-d and 5a ). Collectively these results suggest that long RSH-binding molecules that activate one catalytic function likely trap the enzyme in a restricted conformational state that precludes the opposing catalysis. Discussion The stringent response is a key regulatory pathway involved in virulence and antibiotic resistance. Thus, targeting the stringent response via chemical inhibition of RSH enzymes is an attractive strategy for development of antibacterials that disrupt the (p)ppGpp homeostasis. Here we identified nanobodies that are efficient allosteric modulators of long SYNTH-competent ribosome-associated long RSH Rel and RelA and dissected their mechanism of action. We showed that these nanobodies are effective tools to control the catalytic output of these enzymes in vitro and in vivo precluding bacterial growth under nutrient starvation. Long RSH enzymes are naturally controlled by adaptor proteins that modulate the cellular levels of (p)ppGpp alarmones in addition to the canonical ribosome-dependent pathway ( Fig. 6a-c ). In E. coli , Rsd is a stimulator of the (p)ppGpp-hydrolase activity of SpoT during carbon source starvation 32 ( Fig. 6a ). In absence of c-di-AMP, DarB binds strongly to the SYNTH domain of B. subtilis Rel 48 while analogously, upon phosphorylation, C. crescentus EIIA Ntr binds the RRM domain of Rel which leads to a strong inhibition of hydrolysis 31 and ppGpp accumulation during nitrogen starvation 30 . Both regulators preclude the formation of the τ-state of Rel 18 and inhibit the hydrolase function by restricting the conformational space to an open, SYNTH ON conformation only compatible with (p)ppGpp synthesis 31 , 48 . The combined allosteric effect of favoring synthesis while strongly inhibiting hydrolysis leads to the accumulation of (p)ppGpp. By contrast, NirD binds to both domains of RelA NTD and prevents (p)ppGpp synthesis 36 ( Fig. 6b-c ). Download figure Open in new tab Fig. 6. Naturally occurring allosteric sites of RSH enzyme can be exploited to modulate their activity. Nanobodies such as Nb585 (a) ; Nb94 (b) ; and Nb898 (c) , bind to Rel and RelA mimicking the mode of action of endogenous RSH adaptor proteins that are activated during the cell cycles to modulate the metabolic state. The action of these nanobodies suggests that besides the genetic well-characterised genetic effects on antibiotic tolerance, virulence and survival; chemically targeting RSH enzymes could be a viable pathway to develop drugs active against bacteria. We demonstrated that the nanobodies characterized in this study target key regulatory hotspots in long RSH enzymes, acting as catalytic checkpoints. Nb94 and Nb96 recognize and occlude the antiparallel α-helical motif of the E. coli RelA TGS domain – an element that inspects the CCA end of tRNAs and is essential for RelA’s activation on the starved ribosomal complex 26 , 27 . Thus, selective inactivation the TGS domain with Nb94 and Nb96 countered the intramolecular allosteric signaling that triggers RelA-mediated (p)ppGpp synthesis in response to amino acid starvation. Analogously, Nb898 binds the structural epitope that we have earlier shown in the case of B. subtilis Rel to be recognized by SYNTH-stimulatory factor DarB 48 . Neither Nb898 nor DarB make direct contacts with the active site of the enzyme to enhance (p)ppGpp synthesis. Rather, both modulators counter the formation of the hydrolase-compatible τ-state by preventing the interaction between the RRM domain and the SYNTH domain. This, in turn, leads to a partial opening of SYNTH ( Fig. 4h-i ). Indeed, binding of Nb898 enhances the affinity of the enzyme for ATP, mimicking the stimulatory effect of the 70S ribosome and resembling the genetic truncation of the RRM domain of Rel and RelA that induces slow growth by the toxic accumulation of (p)ppGpp 20 ( Fig. 6c ). The stronger stimulatory effect of Nb898 compared to DarB is likely the result of the higher affinity of Nb898 for Rel (186 nM in the case of Nb898 vs 1.4 μM in the case of DarB 48 ), suggesting that tight-binding molecules targeting this particular region of Rel are likely to induce severe growth arrest. By contrast, Nb585 recognizes a more complex tridimensional epitope that includes both N-terminal catalytic domains. This is reminiscent of the conformational recognition employed by NirD 36 to inhibit RelA. However, from a functional perspective, Nb585 mimics Rsd 32 by stabilizing the close τ-state of the long RSH enzyme to increase the hydrolase activity while inhibiting the (p)ppGpp synthesis. Collectively, functional effects mediated by nanobodies mirror the activity-defining rearrangements stabilized by nanobodies on the cystic fibrosis transmembrane conductance regulator (CFTR) 39 , 52 and GPCRs 53 . Nanobodies targeting extracellular epitopes of proteins are currently being developed as potential drugs for a variety of human diseases, including antimicrobials, however, they are mainly targeting secreted toxins and toxin receptors. While practical applications of intracellular delivery translated to therapeutics are likely to remain a significant challenge, the strong effect shown by the action of the nanobodies on the Galleria infection model suggest the RSH pathway is a viable target to develop novel antibacterials. Rather, small molecule compounds are still the method of choice for intracellular therapeutic targets. Thus, the tridimensional allosteric hotspots revealed using these nanobodies, provides valuable structural insights that can guide drug development and inform future studies aimed at modulating the enzymatic output of long RSHs. Supplementary Figures Download figure Open in new tab SFig. 1. The TGS H432 directly coordinates Nb94 and Nb96. (a) Multiple sequence alignment of the nanobodies Nb94, Nb96 and the control Nb120 (each CDR region is boxed and labeled). Details of the binding interface of Nb94 (b) and Nb96 (c) as predicted by AlphaFold. CDR regions involved in binding are indicated by a black arrow. From the prediction it is clear the H432E substitution will significantly disturb the interface. ITC titrations of Nb94 (d) and Nb96 (e) into RelA Ec H432E . The lack of binding observed strongly supports the predicted mode of binding. Download figure Open in new tab SFig. 2. Nb898 and Nb585 have notably different CDR regions. Sequence comparison of Nb898 and Nb585 shows the strong differences in the observed complementarity determining region (CDR) that defined the contrasting activities of these nanobodies. Each CDR region is labeled in the figure. Download figure Open in new tab SFig. 3. Characterisation of the Rel Ct NTD -Nb898 interface. (a) Heatmaps representing the HDX of Rel Ct NTD (top), Rel Ct NTD -Nb898 complex (center) and ΔHDX (bottom). Residues involved in the binding interface are outlined by a dashed red line. RFU, relative fractional uptake. DarB (b) and Nb898 (c) enhance the (p)ppGpp synthetase activity of Rel. In both cases α13 is the main contact point with additional interactions contributed by α11 and the active site G-loop. Unbiased mFo-DFc electron density map corresponding to the bound APCPP (d) and GDP (e) is shown in dark blue. Download figure Open in new tab SFig. 4. Nb585 stabilises a conformation that resembles the active HD state of Rel. (a) Structural superposition of Rel Ct NTD in the closed conformation predicted in the complex with Nb585 onto the Rel Tt -ppGpp complex. The comparison supports the hypothesis that binding to Nb585 triggers a state that favours hydrolysis (illustrated by the presence of ppGpp in the HD site) and prevents synthesis (the SYNTH active site is largely occluded in this state). (b) Cartoon representation of the Rel Ct NTD -Nb585 complex coloured as in Fig. 5c . The main contact interface is highlighted by a black square. (c) Details of Rel Ct NTD -Nb585 complex interface highlighting key residues involved in binding including D31 of Nb585 (in blue) that forms a slat bridge with R215 of Rel Ct NTD (in gold). SUPPLEMENTARY TABLES View this table: View inline View popup Download powerpoint Supplementary Table 1. Binding parameters determined by Isothermal Titration Calorimetry (ITC). Experimentally determined binding thermodynamic parameters resulting from ITC measurements. N.D. stands for ‘not detectable’. All of the presented titrations are background-subtracted. View this table: View inline View popup Download powerpoint Supplementary Table 2. X-ray data collection and processing. The CC 1/2 criterion was used to determine the resolution range. Values for the outer shell are given in parentheses. Online Methods Multiple sequence alignment Sequences were aligned with MAFFT v7.164b with the L-INS-i strategy 54 and visualised with Jalview 55 . Enzymatic assays Purified protein was incubated in HEPES Polymix buffer (as in Takada et. al., 2020; Tamman et. al., 2020; Takada et. al., 2021; Roghanian et. al., 2021) with HEPES pH 7.5 and 0.5 mM TCEP to a final concentration of 0,25 µM. The Nbs (2.5 µM) or 70S ribosomal complexes (1.5 µM) ( E. coli ) were added prior to a 5 min incubation at room temperature. In case of deacylated tRNA (1 µM) (mix) and pppGpp-mediated (100 µM) stimulation of (p)ppGpp synthesis, these compounds were added after incubation with 70S ribosomal complexes for 5 minutes and incubated for a subsequent 5 minutes at room temperature. When monitoring the ppGpp-synthesis reaction, GDP was added to 1 mM final concentration to the mixture before incubation for 1 min at 40 °C. Synthesis of ppGpp was initiated by the addition of ATP (at 40 °C) to a final concentration of 1 mM. When monitoring the ppGpp-hydrolysis reaction, the reaction mix was incubated at 40 °C for 1 minute before adding the substrate (ppGpp) at a final concentration of 1 mM. The reaction mixtures were incubated in a mixing heat block at 40°C under rigorous shaking at 600 r.p.m. Quenching of the enzymatic reactions was achieved by the addition of formic acid (10 %). The reaction mixes were stored on ice and centrifuged for 1 minute at 4 °C before analysis by means of HPLC. HPLC was performed on a Shimadzu i-series device using an ACE-equivalence C18 HPLC column (3 µM, 30 x4.6 mm, Avantor). Nucleotides were separated by reverse phase applying a gradient (0-100 %) of buffer B (100 mM K phosphate pH 6.4, 5 mM tetrabutyl ammonium bromide (TBAB), 15 % acetonitrile) to buffer A (10 mM K phosphate buffer (pH 6.4, 5 mM TBAB, 0.1 % acetonitrile) at a flowrate of 0.8 mL / minute for 10 minutes. The nucleotides were monitored by absorbance at 254 nm and concentration of GDP and ppGpp was derived from calibration curves using the surface area of the respective peaks. All measurements were analysed using the Labanalysis software. Turnovers were obtained from linear regression of the GDP or ppGpp synthetized in time. All experiments were performed in triplicates. Growth assays on SMG (Serine, Methionine and Glycine) plates The SMG plate assay was performed as described earlier 17 . The strains were grown overnight in liquid M63 44 -glucose minimal medium supplemented with 0.4% glucose, 5 μg/mL thiamine, 100 μg/mL each of L-Arginine, L-Histidine, L-Leucine, L-Threonine and 100 µg/mL ampicillin. Optionally, 0.5 mM IPTG was used to induce nanobody expression and 1 mM each of L-Serine, L-Methionine, and L-Glycine were added to repress growth of RelA-deficient strains. The cultures were adjusted to OD 600 0.5 and 10 fold dilutions were plated on solid M63, SMG and SMG-IPTG plates 43 supplemented with 100 µg/mL ampicillin. Virulence assays in G. mellonella For inoculation, G. mellonella larvae were incubated 1h at 4 °C before injection. Overnight culture of E. coli Ec156 45 transformed with the vector expressing the nanobodies were washed and diluted to a cell density equivalent to 100 CFU in 0.9 % NaCl, 10 µL and were injected in the bottom left proleg of each larva. Twenty larvae were inoculated per strain and incubated at 37 °C in the dark. Viability of the larvae was scored every 12 h. Protein purification Overexpression and purification of E. coli RelA and C. tepidum Rel was performed as described earlier 19 , 56 . To purify Rel Ct NTD and variants, clarified cell lysate of N-terminally His-TEV-tagged proteins was loaded onto a HisTrap HP 1 mL column (GE healthcare) pre-equilibrated with 5 column volumes of the binding buffer (50 mM Hepes pH 7.5, 500 mM KCl, 500 mM NaCl, 10 mM MgCl 2 , 1 mM TCEP and 0.002 % mellitic acid) 1 pastil of protease inhibitors cocktail (Roche)), respectively. A washing step with binding buffer preceded elution of the protein by a linear gradient of 0-500 mM imidazole. Elution fractions were pooled for size exclusion chromatography with a HiLoad 16/600 Superdex 200 PG column (GE healthcare) pre-equilibrated with respective binding buffer. Purified truncates were treated with TEV-protease in a 1:100 molar ratio, overnight, at room temperature to remove the His-tag. The protease and cleaved tag were removed by reverse IMAC using a TALON gravity flow column followed by SEC to the protein buffer. The proteins were concentrated to appropriate concentration using ultrafiltration units with 30 000 Da cut-off (Amicon Ultra, Merck Milipore). Purity of the samples’ preparation was assessed spectrophotometrically and by SDS-PAGE. In all cases nanobodies were produced in E. coli WK6 cells 39 transformed with pMesy4 plasmid carrying the Nb-genes. Cells were grown in TB broth and expression was induced with 500 μM IPTG overnight at 28 °C. Periplasmatic extraction of the nanobodies was done by osmotic shock was achieved as previously described 41 . The His-tagged Nbs were purified using a His-trap Ni-NTA IMAC-column (Cytiva) on an AKTA pure FPLC-system at 8 °C in 25 mM HEPES 7.5; 250 mM NaCl; 1 mM TCEP. Elution of the was performed by applying a gradient of imidazole (0-500 mM). IMAC was followed by buffer exchange on SEC column (Superdex75 increase 10/300 GL, Cytiva) to HEPES 7.5; 250 mM NaCl; 1 mM TCEP. Isothermal affinity calorimetry (ITC) All titrations were performed with an Affinity-ITC (TA instruments) at 15 °C using a constant titration volume of 2μL. Samples were incubated and degassed for 10 minutes prior to the ITC measurement. Nucleotide stocks of Adenosine-5’-[(α,β)-methyleno]triphosphate (ApCpp), ppGpp, pppGpp (Jena Biosciences) and GDP (Sigma Aldrich) of 650–670 mM were diluted in 50 mM HEPES pH 7.5; 500 mM KCl; 500 mM; NaCl; 10 mM MgCl 2 ; 1 mM TCEP; 0.002 % mellitic acid,to a 16-22 fold excess relative to the protein in the ITC cell. The purified RSH-enzymes were concentrated by ultrafiltration (Amicon Ultra, 0.5 ml, 30 kDa, Merck Millipore) at 3,000g at 15 °C to a final concentration of 44–1800 μM. All ITC measurements were performed using a constant stirring rate of 75 r.p.m. All data were processed and analyzed using the NanoAnalyse and Origin software packages, the data is summarized in Supplementary Table S1 . Crystallisation of Rel Ct NTD /Nb898-complex Crystals of Rel Ct NTD in complex with GDP and APCPP were obtained by incubating Rel Ct NTD at 10 mg/mL with 10 mM (final concentration) of the nucleotides. Crystals were obtained in F8 condition of the LMB crystallization screen (Hampton research) at 20 °C using the sitting-drop vapour-diffusion technique with Swissci (MRC) 96-well 2-drop UVP sitting-drop plates. The 200 nL drops of 1:1 crystallization solution/protein sample, were deposited under humidity-controlled conditions using the Mosquito robotic system (TTP Labtech). The drops were equilibrated to 80 μL of the crystallization solution on the plate. To obtain crystals of Rel Ct NTD in complex with Nb898, the nanobody was added in a 5-fold molar excess to purified Rel Ct NTD and incubated for 5 minutes at room temperature. SEC on an Åkta pure FPLC system at 8 °C with a Superdex 200 increase 10/300 GL column (cytiva) equilibrated with the protein buffer at 2% glycerol was performed on the mixture to isolate the protein complex. Formation of the complex was verified on denaturing SDS-PAGE. The protein complex was concentrated by ultrafiltration using a 3000 Da cut-off (Amicon Ultra, Merck Milipore) to 14 mg/mL as estimated by OD 280nm (on a NanoDrop microvolume spectrophotometer, Thermo-Fisher Scientific) using theoretical extinction coefficients. Crystals were obtained in D9 condition of the LMB crystallisation screen (Hampton research) at 20 °C using the sitting-drop vapour-diffusion technique with Swissci (MRC) 96-well 2-drop UVP sitting-drop plates. The 200 nL drops of 1:1 crystallization solution/protein sample, were deposited under humidity-controlled conditions using the Mosquito robotic system (TTP Labtech). The drops were equilibrated to 80 μL of the crystallization solution on the plate. Structure determination X-ray diffraction data of crystals of Rel Ct NTD :Nb898 were collected at the PX2 beamline of the Soleil synchrotron (Gif-sur-Yvette, France) on an Eiger detector. Prior to data collection, crystals the crystals were transferred to a suitable cryoprotectant solution containing 38% of 2-Methyl-1,3-propanediol (MPD) and flash-cooled in liquid nitrogen. The data were processed using XDS suite 37 and scaled with Aimless. The anisotropic analysis of the diffraction data suggested a resolution of 3.15 Å (with diffraction limits of 3.39 Å in a*, 4.99 Å in b* and 2.73 Å in c*). The structure was solved by molecular replacement performed with Phaser 38 using the coordinates of nanobody T4 39 (PDB ID: 6GJS) after removing the CDR regions and the coordinates of Rel Tt NTD . Initial automated model building was performed with Buccaneer 42 which partially completed the Nb898 and further improved with the MR-Rosetta suite from the Phenix package 43 . After several iterations of manual building with Coot 41 and maximum likelihood refinement as implemented in Buster/TNT 44 , the model was refined to R/Rfree of 0.243/0.283. In the case of the Rel Ct NTD :GDP:APCPP complex X-ray diffraction data were collected at the PX2 beamline of the Soleil synchrotron (Gif-sur-Yvette, France) on an Eiger detector. Crystals were cryo-protected in 20%glycerol and 2% DMSO and flash-cooled in liquid nitrogen prior to data collection. The data were processed using XDS suite 37 and scaled with Aimless. Anisotropic analysis of the diffraction data suggested a resolution of 2.65 Å (with diffraction limits of 3.07 Å in a*, 2.65 Å in b* and 2.80 Å in c*). The structure was solved by molecular replacement performed with Phaser 38 using the coordinates of the separated HD and SYNTH domains of Rel Ct NTD (from the Rel Ct NTD -Nb898 complex). Automated model building was performed MR-Rosetta suite from the Phenix package 43 . After several iterations of manual building with Coot 41 and maximum likelihood refinement as implemented in Buster/TNT 44 , the model was refined to R/Rfree of 0.187/0.228. The X-ray data collection and refinement statistics of both datasets are summarized in Supplementary Table S2 . Hydrogen-deuterium exchange mass spectrometry HDX-MS experiments were performed on an HDX platform composed of a Synapt G2-Si mass spectrometer (Waters Corporation) connected to a nanoACQUITY ultraperformance liquid chromatography (UPLC) system, as described 57 . Samples of Rel Ct NTD , Nb898, Nb585 the Rel Ct NTD :Nb898 and Rel Ct NTD :Nb585 complexes were prepared at a concentration of 50 to 70 μM. For each experiment, 5 μl of sample was incubated for 0.5, 5, or 60 min in 95 μl of labeling buffer L [50 mM HEPES, 500 mM KCl, 500 mM NaCl, 2 mM MgCl 2 , 1 mM TCEP, and 0.002% mellitic acid (pH 7.5)] at 20 °C. The nondeuterated reference points were prepared by replacing buffer L by equilibration buffer E [50 mM Hepes, 500 mM KCl, 500 mM NaCl, 2 mM MgCl 2 , 1 mM TCEP, and 0.002% mellitic acid (pH 7.5)]. After labeling, the samples are quenched by mixing with 100 μl of prechilled quench buffer Q [1.2% formic acid (pH 2.4)]. Seventy microliters of the quenched samples were directly transferred to the Enzymate BEH Pepsin Column (Waters Corporation) at 200 μl/min and at 20 °C with a pressure of 8.5 kpsi. Peptic peptides were trapped for 3 min on an ACQUITY UPLC BEH C18 VanGuard precolumn (Waters Corporation) at a flow rate of 200 μl/min in water [0.1% formic acid in HPLC water (pH 2.5)] before being eluted to an ACQUITY UPLC BEH C18 column for chromatographic separation. Separation was performed with a linear gradient buffer (7 to 40% gradient of 0.1% formic acid in acetonitrile) at a flow rate of 40 μl/min. Identification of peptides and deuteration uptake analysis was performed on the Synapt G2Si in (ESI+) - HDMSE mode (Waters Corporation). Leucine enkephalin was applied for mass accuracy correction, and sodium iodide was used as calibration for the mass spectrometer. HDMSE data were collected by a 20- to 30-V transfer collision energy ramp. The pepsin column was washed between injections using pepsin wash buffer [1.5 M Gu-HCl, 4% (v/v) methanol, and 0.8% (v/v) formic acid]. A blank run was performed between each sample to prevent significant peptide carryover. Optimized peptide identification and peptide coverage for all samples were performed from undeuterated controls (five replicates). All deuterium time points were performed in triplicate. Acknowledgments This work was financially supported by the Fonds National de Recherche Scientifique (CDR J.0065.23F; PDR T.0090.22), ERC (CoG DiStRes, n° 864311), the Fonds Jean Brachet, the Fondation Van Buuren and FNRS WELBIO ADV X.1520.24F, INSTRUCT (PID: 4098), all to A.G-P. C.M. was supported by Fonds de la Recherche Scientifique (MIS grant F.45322.22). K.C.W. and K.V.N. were supported by FRIA. The authors acknowledge the use of the PROXIMA-2A beamline at the Soleil synchrotron (Gif-sur-Yvette, France) and I24 (Diamond Light Source synchrotron Oxfordshire UK). 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Share Nanobody-mediated modulation of long RSH enzymes Rel and RelA catalysis by restriction of their conformational landscape Katleen Van Nerom , Andres Ainelo , Kyo Coppieters ’t Wallant , Ariel Talavera-Perez , Dannele Echemendia-Blanco , Sarah Peeters , Brahim El Khalfaoui Oulali , Hedvig Tamman , Tatsuaki Kurata , Mohammad Roghanian , Chloé Martens , Els Pardon , Jan Steyaert , Vasili Hauryliuk , Abel Garcia-Pino bioRxiv 2025.08.07.669093; doi: https://doi.org/10.1101/2025.08.07.669093 Share This Article: Copy Citation Tools Nanobody-mediated modulation of long RSH enzymes Rel and RelA catalysis by restriction of their conformational landscape Katleen Van Nerom , Andres Ainelo , Kyo Coppieters ’t Wallant , Ariel Talavera-Perez , Dannele Echemendia-Blanco , Sarah Peeters , Brahim El Khalfaoui Oulali , Hedvig Tamman , Tatsuaki Kurata , Mohammad Roghanian , Chloé Martens , Els Pardon , Jan Steyaert , Vasili Hauryliuk , Abel Garcia-Pino bioRxiv 2025.08.07.669093; doi: https://doi.org/10.1101/2025.08.07.669093 Citation Manager Formats BibTeX Bookends EasyBib EndNote (tagged) EndNote 8 (xml) Medlars Mendeley Papers RefWorks Tagged Ref Manager RIS Zotero Tweet Widget Facebook Like Google Plus One Subject Area Microbiology Subject Areas All Articles Animal Behavior and Cognition (7637) Biochemistry (17705) Bioengineering (13899) Bioinformatics (41970) Biophysics (21463) Cancer Biology (18605) Cell Biology (25526) Clinical Trials (138) Developmental Biology (13385) Ecology (19911) Epidemiology (2067) Evolutionary Biology (24329) Genetics (15615) Genomics (22514) Immunology (17743) Microbiology (40424) Molecular Biology (17194) Neuroscience (88650) Paleontology (667) Pathology (2835) Pharmacology and Toxicology (4827) Physiology (7648) Plant Biology (15160) Scientific Communication and Education (2046) Synthetic Biology (4302) Systems Biology (9825) Zoology (2271)