Mechanistic insights into the allosteric regulation of cell wall hydrolase RipA inMycobacterium tuberculosis

Mechanistic insights into the allosteric regulation of cell wall hydrolase RipA in Mycobacterium tuberculosis | 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 j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (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];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Mechanistic insights into the allosteric regulation of cell wall hydrolase RipA in Mycobacterium tuberculosis View ORCID Profile Giacomo Carloni , View ORCID Profile Quentin Gaday , View ORCID Profile Julienne Petit , View ORCID Profile Mariano Martinez , View ORCID Profile Daniela Megrian , View ORCID Profile Adrià Sogues , View ORCID Profile Mathilde Ben Assaya , Marcell Kakonyi , View ORCID Profile Ahmed Haouz , View ORCID Profile Pedro M. Alzari , View ORCID Profile Anne Marie Wehenkel doi: https://doi.org/10.1101/2025.06.28.662095 Giacomo Carloni 1 Institut Pasteur, Université Paris Cité, CNRS UMR 3528, Bacterial Cell Cycle Mechanisms Unit , F-75015 Paris, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Giacomo Carloni Quentin Gaday 2 Institut Pasteur, Université Paris Cité, CNRS UMR 3528, Structural Microbiology Unit , F-75015 Paris, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Quentin Gaday Julienne Petit 1 Institut Pasteur, Université Paris Cité, CNRS UMR 3528, Bacterial Cell Cycle Mechanisms Unit , F-75015 Paris, France 2 Institut Pasteur, Université Paris Cité, CNRS UMR 3528, Structural Microbiology Unit , F-75015 Paris, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Julienne Petit Mariano Martinez 1 Institut Pasteur, Université Paris Cité, CNRS UMR 3528, Bacterial Cell Cycle Mechanisms Unit , F-75015 Paris, France 2 Institut Pasteur, Université Paris Cité, CNRS UMR 3528, Structural Microbiology Unit , F-75015 Paris, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Mariano Martinez Daniela Megrian 3 Institut Pasteur de Montevideo, Bioinformatics Unit , 11200 Montevideo, Uruguay Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Daniela Megrian Adrià Sogues 2 Institut Pasteur, Université Paris Cité, CNRS UMR 3528, Structural Microbiology Unit , F-75015 Paris, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Adrià Sogues Mathilde Ben Assaya 2 Institut Pasteur, Université Paris Cité, CNRS UMR 3528, Structural Microbiology Unit , F-75015 Paris, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Mathilde Ben Assaya Marcell Kakonyi 1 Institut Pasteur, Université Paris Cité, CNRS UMR 3528, Bacterial Cell Cycle Mechanisms Unit , F-75015 Paris, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site Ahmed Haouz 4 Institut Pasteur, Plate-forme de cristallographie – C2RT, CNRS UMR 3528, Université Paris Cité , F-75015 Paris, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Ahmed Haouz Pedro M. Alzari 2 Institut Pasteur, Université Paris Cité, CNRS UMR 3528, Structural Microbiology Unit , F-75015 Paris, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Pedro M. Alzari For correspondence: pedro.alzari{at}pasteur.fr anne-marie.wehenkel{at}pasteur.fr Anne Marie Wehenkel 1 Institut Pasteur, Université Paris Cité, CNRS UMR 3528, Bacterial Cell Cycle Mechanisms Unit , F-75015 Paris, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Anne Marie Wehenkel For correspondence: pedro.alzari{at}pasteur.fr anne-marie.wehenkel{at}pasteur.fr Abstract Full Text Info/History Metrics Preview PDF ABSTRACT D,L-endopeptidase RipA is the major PG hydrolase required for daughter cell separation in Mycobacterium tuberculosis ( Mtb ), as RipA defects severely hinder cell division and increase antibiotic vulnerability. Despite extensive studies, the mechanisms governing Mtb RipA regulation remain controversial and poorly understood. Here, we report an integrative structural and functional analysis of the SteAB system, a regulatory complex that has been shown to modulate cell separation in the model organism Corynebacterium glutamicum ( Cglu ) and is conserved across Mycobacteriales . Although Mtb SteB was previously proposed to act as a mycobacterial outer membrane copper transporter, the crystal structures of the homodimeric protein, alone and in complex with the RipA coiled-coil (CC) domain, rule out this hypothesis. Instead, the high-affinity SteB-RipA interaction, together with computational and biophysical data, strongly supports the role of SteB as a direct RipA activator that releases enzyme autoinhibition upon complex formation. In addition, crystallographic characterization of the cytoplasmic core of SteA revealed a homodimeric organization harboring a conserved functional pocket similar to the phosphonucleotide-binding site of thiamine pyrophosphokinase. These data, coupled with the in vivo phenotypic analysis of a steAB knockout mutant of Cglu , support a model in which the transmembrane SteAB heterotetramer, driven by cytoplasmic ligand binding, orchestrates the productive periplasmic positioning of RipA, leading to PG hydrolysis activation. These findings shed new light on the regulation of mycobacterial cell wall remodeling, with implications for understanding Mtb pathogenesis and identifying novel antimicrobial targets. INTRODUCTION Peptidoglycan (PG), the main component of the bacterial cell wall, consists of glycan strands of two alternating sugar molecules, N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM), cross-linked by short pentapeptide stems. This cage-like polymer surrounds the plasma membrane, confers cell shape and protection against osmotic disruption, and is continually remodeled during the cell cycle through the coordinated action of different PG hydrolases and synthetases ( 1 ). In polar-growing bacteria, such as Mycobacteriales , which do not undergo septal constriction during cytokinesis, the PG mesh forms a continuous mechanical linkage between the progeny cells at the nascent division plane. At the end of cell division, this linkage must be released to allow for daughter cell separation (V-snapping) ( 2 ). This crucial task is performed by an array of PG hydrolases, whose tight regulation ensures that cell division occurs in a timely and organized manner, preventing uncontrolled cell wall degradation that can lead to morphological defects or bacterial lysis. These defective cell division phenotypes are often accompanied by increased membrane permeability and antibiotic susceptibility, making PG hydrolases attractive targets for the development of novel antimicrobial agents. In the human pathogen Mycobacterium tuberculosis ( Mtb ), D-L endopeptidase RipA (Rv1477) is the major PG hydrolase involved in cell separation ( 3 – 5 ). Although there are other genes encoding PG hydrolases in Mtb , RipA is the only endopeptidase whose depletion induces severe morphological defects and in vivo reduced infectivity, emphasizing its importance for proper cell separation and integrity. RipA is a member of a conserved clade of the NlpC/P60 enzyme superfamily that cleaves stem peptide bridges within the PG mesh, and has been shown to be important for cell separation in other Mycobacteriales species ( 2 , 6 – 8 ). Despite its essential role in Mtb cell division, limited and controversial information is available regarding the underlying regulatory mechanism(s) of this process. RipA interacts in vivo with other cell division proteins, notably the penicillin-binding protein PBP1 and resuscitation-promoting factor RpfB ( 9 , 10 ), which may be involved in enzyme regulation through protein-protein interactions. Moreover, truncated RipA species were found in cell wall compartments and culture filtrates, suggesting that RipA is proteolytically processed in vivo ( 4 ), and the protease MarP was reported to hydrolyze RipA during acid stress ( 11 ). More recently, it has been shown that the periplasmic membrane-associated protein SteB binds to and activates full-length RipA in Corynebacterium glutamicum (Cglu) by dissociating the intramolecular complex between the catalytic and the EnvC-like coiled-coil (CC) domains ( 12 ). SteB is part of a septal transmembrane complex with the cytosolic membrane protein SteA, and inactivation of either steA or steB (adjacent genes in the same operon) phenocopies RipA inactivation in terms of ethambutol hypersensitivity and cell wall defects ( 13 ). These findings demonstrated that the SteAB complex is part of an FtsEX-independent regulatory system for cell wall degradation mediated by RipA ( 12 ). However, the underlying molecular mechanisms remain unclear. Although the steAB operon is conserved in Mycobacteriales , the SteB homolog in Mtb (Rv1698) has previously been described as a putative channel-forming protein involved in copper transport ( 14 , 15 ), and therefore renamed MctB (for Mycobacterial c opper transport protein B ). The Mtb SteA homolog (Rv1697) is an uncharacterized hypothetical protein annotated as a thiamin pyrophosphokinase that is essential for the in vitro growth of Mtb H37Rv ( 16 – 19 ). For clarity, we will refer to these proteins respectively as Mt SteA (Rv1697) and Mt SteB (Rv1698) in the rest of this manuscript. Here, we report an integrative structural analysis of the SteAB system in Mtb. The crystal structures of homodimeric Mt SteB, both alone and in complex with the CC domain of RipA, redefine the role of the protein as a regulator of RipA in Mtb , ruling out a putative direct role in copper transport. These findings, together with the crystallographic characterization of homodimeric Mt SteA, the structural analysis of the Mtb SteA/SteB/RipA ternary complex and the phenotypic characterization of a SteAB-defective Cglu mutant strain, put forward a model in which, upon cytoplasmic ligand recognition and CC-mediated intracellular signal transduction, formation of the transmembrane SteAB heterotetramer allows for the productive positioning of RipA to activate PG hydrolysis in the periplasm. Elucidating the intricacies of RipA function enhances our understanding of Mtb pathogenesis and opens new avenues for the design of innovative strategies to combat tuberculosis. RESULTS The structures of M. tuberculosis SteB, alone and in complex with the RipA coiled-coil domain Mt SteB is a 33 kDa membrane-bound protein (314 amino acids) with a single N-terminal transmembrane (TM) segment. For structural studies, we produced the soluble protein devoid of its TM domain ( Mt SteB ΔTM , residues 38-314) and determined its crystal structure at 2 Å resolution. The protein is a homodimer with a central CC dimerization domain (residues 41-76 from each protomer), surrounded on either side by the respective C-terminal globular cores (residues 77-314) ( Fig. 1A ). The monomeric core, which displayed a (β/α) topology ( Fig. S1A ), is similar to that of the homologous SteB from Cglu (( 12 ), pdb code 8AU6), with an RMSD of 1.019 Å for 168 Cα equivalent positions ( Fig. S1B ). A major difference, however, is that Cg SteB crystallized as a monomer, whereas Mt SteB is a homodimer. The dimeric conformation is mediated by the intermolecular parallel CC formed between the N-terminal α-helices of each protomer ( Fig. 1B ). The CC was further stabilized by interactions of the helix tip from one protomer with the C-terminus of the second protomer ( Fig. S1C ). Interestingly, in Cg SteB this C-terminal region (residues 296-314) is missing from the construct ( 12 ) and could explain why it crystallized as a monomer. Close inspection of the residues involved in CC formation revealed that the heptad repeat pattern is conserved in SteB homologs from other Mycobacteriales ( Fig. 1C ). Despite the lower conservation of the heptad repeat in the Cglu protein, the AlphaFold (AF) model of Cg SteB predicted the same CC-mediated dimerization mode as seen in Mt SteB crystals, thus supporting the SteB homodimer as the functional unit. In the full-length protein, the TM helix immediately precedes the CC helix in full-length SteB ( Fig. 1C ), and may thus play a role in stabilizing (or modifying) the CC domain structure, suggesting a possible mechanism for conformational signal transduction. Download figure Open in new tab Figure 1. Overall structure of Mt SteB. (A) Cartoon representation of the SteB homodimer, with each monomer in pink and blue, respectively. (B) Detailed view of the N-terminal coiled-coil (CC), showing the residue side-chains at the a and d positions of the heptad repeats. (C) Alignment of the SteB CC region from selected species of Mycobacteriales . The TM region and the ( a-g ) heptad repeat positions are shown below the alignment, with the a and d positions shown in orange and blue respectively. PCCP indicates the probability of parallel CC formation ( 68 ). (D) BLI binding curves of immobilized Mt RipA CC against Mt SteB. (E) Overall view of the Mt SteB ΔTM crystal structure in complex with Mt RipA CC . The inset shows a ribbon representation of the interface, in which residues involved in hydrogen bonding interactions are labeled. (F) BLI binding curves of immobilized Mt RipA CC against Mt RipA CAT . (G) The analysis of residue conservation (ConSurf) in all Mycobacteriales identifies two non-overlapping regions in the N-terminal CC of RipA (shown here in the same orientation as in panel E) that interact with the C-terminal catalytic domain and the activator protein SteB, respectively. Given the structural similarity between the SteB homologs from Mtb and Cglu , it is likely that Mt SteB, like Cg SteB, plays a direct regulatory role for RipA, rather than the initially proposed role in copper transport ( 15 ). Supporting this hypothesis, we observed that Mt SteB ΔTM interacted with the CC domain (residues 40-240) of RipA ( Mt RipA CC ) with an apparent dissociation constant Kd of 42.6 ± 4.4 μM ( Figs. 1D and Fig. S2 ), as determined by bio-layer interferometry (BLI). Furthermore, we crystallized the Mt SteB ΔTM – Mt RipA CC complex and solved its 3D structure at 2.2 Å resolution ( Table S1 ). The structure revealed a 2:2 heterotetramer ( Fig. 1E ), where the Mt SteB homodimer observed in the apo structure was retained. Mt RipA CC folds into an antiparallel helical hairpin, with helices α1 (residues 46-120) and α2 (residues 138-238). The two Mt RipA CC molecules in the complex aligned parallel to each other and were roughly perpendicular to the membrane plane. Protein-protein association is mediated by hydrogen bonding and hydrophobic interactions between helix α2 of Mt RipA CC and the loop connecting helices α3-α4 (residues 139-153) of Mt SteB ( Fig. 1E inset). Interestingly, the α3-α4 loop, which is well defined in the structure of the complex, was disordered in the apo Mt SteB structure and was not visible in the electron density map ( Fig. S3 ), indicating an induced-fit mechanism for RipA recruitment. Mt RipA is autoinhibited via its N-terminal CC domain The SteB-RipA complex in Mtb has an interface similar to that previously predicted for the homologous complex in Cglu and was validated by site-directed mutagenesis ( 12 ). However, full-length Cg RipA crystallized in an autoinhibited form, with its N-terminal CC domain bound to and blocking the catalytic site ( 12 ). In contrast, two independent crystal structures of the Mt RipA catalytic domain ( Mt RipA CAT ), a form that lacks the N-terminal CC domain but includes 16 residues immediately preceding the catalytic domain (residues 264-279) ( 20 , 21 ), showed that the active site was blocked by this region ( Fig. S4A ). These structures lent credit to the hypothesis that Mt RipA is a zymogen and suggested that its N-terminal CC domain may have a different function ( 22 ). To investigate this apparent discrepancy, we predicted structural models of truncated and full-length MtRipA forms using AlphaFold (AF). The active site interactions observed in the previous crystal structures ( 20 , 21 ) were only observed for constructs lacking the N-terminal CC domain. However, in the full-length protein the active site was predicted to bind the CC ( Fig. S4B ), to form a complex similar to that observed in the crystal structure of Cg RipA ( 12 ). To experimentally validate this interaction, we produced separate constructs for Mt RipA CC (residues 40-240) and Mt RipA CAT (residues 261-472) and measured their binding affinity using BLI. The apparent Kd value obtained (6.8 ± 1.4 μM, Fig. 1F and Fig. S4C ) strongly supports the CC-mediated autoinhibitory model. In summary, the above findings demonstrate that the Mt RipA CC domain uses two well-conserved, non-overlapping regions to bind respectively its own catalytic domain and the activator protein Mt SteB ( Fig. 1G ). Structural characterization of SteA steB ( rv1698 ) and steA ( rv1697 ) are part of the same conserved operon in Mycobacteriales . The corresponding proteins were described as protein partners in Cglu ( 13 ) and are encoded by a single fused gene in some species. Mt SteA is a 43 kDa protein (393 amino acid residues) that consists of an N-terminal cytoplasmic core followed by a TM helix (residues 344-366, Mt SteA numbering) and a C-terminal amphipathic helix exposed on the periplasmic side (residues 371-393). For structural studies we produced a truncated soluble construct (residues 12-344, Mt SteA ΔTM ) that forms a dimer in solution ( Fig. S5A ) and determined its crystal structure at 2 Å resolution ( Table S1 ). The Mt SteA ΔTM homodimer exhibits an overall ‘paper boat’ shape ( Fig. 2A ), in which the monomers fold into a central C-terminal dimerization domain (the sail) connected through a long (31 residues) α-helix (the hull) to the distal N-terminal globular domains. Protein dimerization buries a largely hydrophobic surface of 2070 Å 2 from the central linker helix and the C-terminal domain of each monomer (accounting for 13% of the total surface area) and is stabilized by several intermolecular hydrogen bonds and two salt bridges involving residues Arg72 and Asp166 from each protomer. Download figure Open in new tab Figure 2. Structure of Mt SteA. (A) Side and top views of the Mt SteA homodimer, with the monomers shown in yellow and green, respectively. (B) Protein dimerization results in an intermolecular 14-stranded Δ-sheet (shown in red) formed by the two C-terminal domains. (C) Molecular surface of Mt SteA colored by electrostatic charges. (D) Conservation pattern of all Mt SteA basic residues (Arg or Lys) in Mycobacteriales SteA homologs. Membrane-facing residue positions, represented by full dots, are generally more conserved. The N-terminal globular domain (residues 12-127) consists of an external four-stranded antiparallel β-sheet orthogonally packed against a four-stranded parallel β-sheet, covered in turn by three helices ( Fig. 2A and Fig. S5B ). This three-layer (β/β/⍺) fold resembles that of the swivelling phosphohistidine domain of phosphoenolpyruvate-transferring enzymes (IPR036637) ( Fig. S6 ), although the catalytic histidine is missing in SteA. This structural domain makes only a few intramolecular contacts with the central Mt SteA protein core and displays a high intrinsic flexibility. This was confirmed by the crystal structure of the closely similar SteA homolog from Cglu ( Cg SteA), which was determined at 2.05 Å resolution ( Table S1 ) and contained eight independent molecules in the asymmetric unit. In all molecules, the N-terminal domain exhibited high B factors ( Fig. S7A ), and the overall superposition of the Cg SteA and Mt SteA monomer structures revealed a wide range of movement of the N-terminal domain ( Fig. S7B and Supplementary Movie SM1). The C-terminal domain of Mt SteA (residues 161-342) is responsible for dimerization and forms the central core of the homodimer ( Fig. 2A ). The domain displayed an (⍺/β) 7 ovoid fold ( Fig. S5B ) in which the parallel β-sheet extends, upon dimerization, into a 14-stranded twisted β-sheet flanked by α-helices on both sides ( Fig. 2B ). At the C-terminus of the dimeric cytoplasmic core, the last α-helices from each protomer immediately preceding the TM helices run parallel to and interact with each other, defining the orientation of the protein with respect to the membrane. In agreement with this hypothesis, the Coulombic electrostatic potential of the protein surface reveals a positively charged membrane-proximal surface ( Fig. 2C ). This surface includes an array of basic residues (Arg/Lys) that are highly conserved in SteA homologs from Mycobacteriales ( Fig. 2D ) and could interact with negatively charged membrane phospholipids. Sequence conservation analysis of SteA reveals two clearly distinct patches of conserved residues on the molecular surface of the monomer ( Fig. 3A ). The larger patch matches the dimerization interface, indicating a conserved homodimerization mode across species. The second patch, on the opposite side of the monomer, corresponds to a protein pocket exposed to the solvent, which may be associated with a ligand-binding site. Structural homology searches using DALI ( 23 ) revealed that the C-terminal domain is similar to the N-terminal ATP-binding domain of thiamine pyrophosphokinase (TPPK, ( 24 )) (rmsd of 1.0 Å for 40 equivalent Cα positions) and, to a lesser extent, to sialyl-tranferase CstII from Campylobacter jejuni (CstII, ( 25 )) (rmsd of 1.0 Å for 33 equivalent Cα positions). Although SteA homologs are annotated in sequence databases as putative TPPKs because of this partial similarity, the putative thiamine-binding site is completely missing in SteA homologs due to a different quaternary organization of the protein. Instead, the structural superposition of Mt SteA with both TPPK and CstII demonstrated that the conserved SteA pocket corresponds to the phosphonucleotide-binding sites for AMP and CMP, respectively ( Fig. 3B ), strongly arguing for a functional SteA binding site. In our hands, analyzing the binding of various phosphonucleotides to the soluble construct of Mt SteA using nano differential scanning fluorimetry (nanoDSF) approaches led to weak, non-specific protein destabilization, which proved inconclusive ( Fig. S8 ). In contrast, a similar investigation on the closely related Cg SteA homolog revealed that the addition of GDP or UDP, but not ADP or triphosphate nucleotides, significantly stabilized the protein ( Fig. 3C ). Although preliminary, these findings suggest that SteA functions as a specific phosphonucleotide-binding protein and imply that cytoplasmic ligand binding and/or hydrolysis might serve as the potential driving force for conformational signal transduction in RipA regulation. Download figure Open in new tab Figure 3. SteA is a putative phosphonucleotide-binding protein. (A) Mapping of conserved residues on the molecular surface of the protein, as calculated by Consurf ( 69 ). The large, conserved patch in the left panel corresponds to the dimerization interface, and the smaller conserved patch in the right panel corresponds to a putative ligand-binding site . (B) The putative Mt SteA binding pocket (left) matches the phosphonucleotide-binding sites of TPPK in complex with AMP (PDB 2f17, center) and CstII in complex with CMP (PDB 1ro7, right). The bound ligands are shown in stick representation. (C) NanoDSF binding assay of different phosphonucleotides to Cg SteA. Coordinated and synergistic action of SteA and SteB in cell wall integrity To understand the role of SteA and SteB in vivo , we used homologous recombination ( 26 ) to generate a ΔsteAB depletion strain in C. glutamicum ATCC13032 ( Cglu_ ΔsteAB ). As steA and steB are the last genes of a predicted 8-gene operon ( 27 ) that contains several putative transcription start sites, one of which overlaps with the steA coding sequence ( Fig. S9A ), we hypothesized that the removal of steA would also silence steB through polar effects. The Cglu _ Δ steAB strain resulted in elongated, multiseptal cells ( Fig. 4A-C ) as previously described for the mutants of the individual genes ( 13 ). We found that the ectopic expression of Cg SteA alone was not sufficient to restore the wild-type Cglu phenotype and only expression of both Cg SteA and Cg SteB fully complemented the mutant strain ( Fig. 4A-C ). We further confirmed the absence of Cg SteB in Cglu _Δ steAB by anti-SteB Western Blot ( Fig. S9B ). Download figure Open in new tab Figure 4. SteA/B depletion and complementation in C. glutamicum . (A) Representative images in Phase contrast (left) and membrane staining (Nile red, right) for the indicated strains. Scale bars = 5 μm. (B) Violin plots showing the distribution of cell length (Cohen’s d , from top to bottom: (***, d = 1,65, p ∼ 0), (ns, d = 0,29, p = 2,44e-19), (***, d = 1,96, p ∼ 0)); the whiskers indicate the 25th to the 75th percentile, and the middle line the median. (C) Frequency histogram showing the number of septa per cell for the different strains, calculated from 3 independent experiments for each strain. Bars represent the mean ± SD. (D) Ethambutol sensitivity assay. BHI overnight cultures of the indicated strains were normalized to an OD600 of 0.5, serially diluted 10-fold, and spotted onto BHI agar medium with or without 1 µg/mL carbenicillin or 0.3 µg/mL ethambutol. As endopeptidase defects have been associated with an increased sensitivity to cell-wall targeting antibiotics such as β-lactams in Mycobacteriales ( 3 , 7 ), we tested the Cglu _Δ steAB mutant strain for carbenicillin sensitivity. The mutant displayed antibiotic sensitivity, and wild-type-like carbenicillin resistance could only be restored upon ectopic expression of both Cg SteA and Cg SteB, but not when expressing the individual proteins ( Fig. 4D ). To investigate if this phenotype could be due to the SteA/B-mediated control of RipA, we produced the Leu146-Arg point mutant of Cg SteB ( Cglu_ SteB L146R ), that was previously shown to abolish the SteB-RipA interaction in vitro ( 12 ). We observed that the ectopic expression of Cg SteA/ Cg SteB, but not that of Cg SteA/ Cg SteB L146R , restored wild-type-like carbenicillin tolerance in the Cglu _Δ steAB strain, even if SteB L146R ectopic expression levels were comparable to wild-type Cg SteB ( Fig. S9B ). As steA and steB were first identified in a transposon mutagenesis screening to identify genes associated with sensitivity to ethambutol ( 13 ), we also observed a hypersusceptibility to ethambutol in the Cglu_ΔsteAB strain ( Fig. 4D ). Surprisingly, however, the ectopic expression of Cg SteA alone was sufficient to restore wild-type-like ethambutol tolerance ( Fig. 4D ), suggesting that SteA may have an additional SteB-independent role, possibly mediated by protein-protein interactions with other septal components of the divisome. These findings in Cglu could explain why Mt SteA, but not Mt SteB, is essential for Mtb growth ( 16 – 19 ). A CC-mediated mechanotransduction mechanism in RipA activation Previous research in Cglu showed that the two transmembrane proteins, SteA and SteB, form a complex that localizes to the cytokinetic ring ( 13 ). The AF prediction of this complex in Mtb revealed a ( Mt SteA/ Mt SteB) 2 heterotetramer ( Fig. S10 ). On either side of the plasma membrane, SteA and SteB display the same homodimeric arrangement of their soluble cores as seen in their respective crystal structures, and interact with each other through their TM regions, assembled into a four-helix bundle. Integrating this model with the crystal structure of the MtSteB-MtRipACC complex described above provides a three-dimensional illustration of the ternary complex involving the SteAB regulatory system and RipA hydrolase ( Fig. 5A ). In this model, both the SteB CC and the attached RipA CC are oriented at approximately right angles to the membrane plane, positioning the NlpC/P60 catalytic domain deep within the periplasmic space. This configuration enables the catalytic domain to physically interact with the PG substrate and would therefore correspond to the active enzyme state. Furthermore, for the NlpC/P60 domain to perform its role, it must be capable of deeply penetrating the porous PG sacculus, which has an average width of approximately 70 Å in Mtb ( Fig. 5A ). This is achievable because of the relatively long linker (∼100 residues) that connects the NlpC/P60 domain to the Mt SteB-bound CC in Mt RipA. Although this linker length is well-conserved among mycobacterial RipA homologs, it is consistently longer in corynebacterial homologs ( Fig. 5B ), matching a thicker PG layer in these species (∼150-180 Å in Cglu , Fig. 5A ). Download figure Open in new tab Figure 5. Mt SteAB-mediated regulation of Mt RipA. (A) Proposed model of the ternary complex of SteA, SteB and RipA in an active conformation. The NlpC/P60 catalytic domain (orange) is connected to the N-terminal coiled-coil domain (white) by a flexible linker (dotted line). This connecting linker has different lengths in Mtb (shown at left) and Cglu (shown at right), which correlates with the approximate thickness of the PG layer in these bacteria ( 70 , 71 ). The inset shows the predicted TM 4-helix bundle. The inactive state (not shown) might be associated to a SteB conformation with a modified or disrupted CC, as seen for instance for the monomeric Cg SteB crystal structure ( 12 ), which would preclude the NlpC/P60 catalytic domain from reaching the PG substrate. ( B ) Distribution of connecting linker lengths in RipA homologs from Mycobacteria (cyan) and Corynebacteria (pink). DISCUSSION The structural characterization of the SteAB complex and its interactions with RipA provides important functional insights into the mechanism by which this system controls daughter cell separation in Mtb . The cytoplasmic domain of Cg SteA can bind GDP- or UDP-containing molecules, pointing to a possible source of power to trigger SteAB-mediated RipA activation. Although further work is required to identify the specific ligand, different intermediate metabolites and recycling molecules from cell wall synthesis do contain this class of phosphonucleotide moieties ( 28 , 29 ). This suggests that the system may be able to sense the status of the cell wall to coordinate cytokinesis. Upon cytoplasmic ligand binding (and/or hydrolysis), the initial activation signal is transmitted through the SteAB TM helical bundle to modulate periplasmic RipA recruitment and activation by directly affecting the productive positioning of the catalytic domain for PG hydrolysis. Both four-helix bundles and two-helical CCs are ubiquitous sensory modules involved in bacterial signal transduction ( 30 ). Indeed, the proposed SteAB mechanism is reminiscent of those described for bacterial TM histidine kinases, where a conformational signal transmitted along the membrane-connecting two-helical coiled-coil serves as a switching mechanism to control enzyme activity ( 31 – 33 ). As both SteA and SteB have been reported to interact with other divisome proteins ( 34 , 35 ), we cannot exclude the possibility that additional septal proteins – yet to be identified – could transiently interact with the TM SteAB helical bundle or the cytoplasmic domain of SteA to fine-tune signal transduction. It was previously proposed that Mt SteB (MctB) could be an outer membrane porine involved in copper transport ( 15 ). Shortly thereafter, however, the same researchers suggested that the protein might be anchored to the inner membrane and could fulfill a more pleiotropic role ( 36 , 37 ). Our findings confirm and expand on the second hypothesis. A primary role of Mt SteB in the control of daughter cell separation can explain the severe growth defects observed for a ρ steB Mycobacterium smegmatis mutant strain or the reduced virulence of a SteB-deficient Mtb mutant strain in mice and guinea pigs, originally attributed to copper toxicity ( 15 ). Several lines of evidence support the involvement of the SteAB system in cell division: both SteA and SteB were identified as direct or indirect interaction partners of two core divisome proteins, FtsB and FtsQ, in Mycobacteria ( 34 , 38 ); the absence of the cell division membrane protein PerM in a Mtb strain resulted in the increased transcription of the two genes, steA and steB ( 39 ); a SteA-deficient Mycobacterium abscessus strain displayed a multi-septa phenotype and higher antibiotic susceptibility ( 40 ); and transposon insertion mutants of steA in Mycobacterium avium led to cell wall modifications and reduced multidrug resistance ( 41 ). In addition to the SteAB system, the ABC transporter-like FtsEX is also involved in the regulation of PG hydrolysis in Mtb ( 42 ). FtsEX controls the action of RipC, another NlpC/P60 hydrolase with a domain organization similar to RipA but whose physiological role remains unclear ( 43 , 44 ). The recent cryo-EM structure of the FtsEX-RipC complex ( 45 ) revealed that RipC binds in an inclined manner and, like RipA, is self-inhibited by its own N-terminal CC domain ( Fig. 6 ). A common feature of these two systems is that enzyme activation relies on the productive periplasmic positioning of the NlpC/P60 domain, as the activation of RipC by FtsEX causes its CC domain to tilt towards the PG layer ( 45 ), favoring the physical interaction of the catalytic domain with its substrate. However, these two systems differ markedly in their overall architectures, mechanisms of action and biological roles. FtsEX belongs to subfamily VII of ABC transporters and uses ATP hydrolysis as the power source to control PG hydrolysis. In contrast, SteAB presents a novel architecture, with a cytoplasmic moiety partially resembling TPPKs that binds phosphonucleotide-containing molecules but not ATP, and a periplasmic moiety that constitutively recruits RipA. Download figure Open in new tab Fig. 6. Two regulatory systems of PG hydrolysis in Mtb . Overall views of the SteAB-RipA complex (left panel) and the FtsEX-RipC complex (right panel, PDB 8JIA, ( 45 )). The NlpC/P60 catalytic domains (shown in orange) are shown in their autoinhibited state, with their active sites bound to their respective N-terminal coiled-coil domains (in white). In both cases, phosphonucleotide binding to the cytoplasmic domain (SteA or FtsE) would draw the respective NlpC/P60 domains towards the PG layer, where the physical interaction with substrate and/or a conformational signal propagated across the membrane would release the catalytic domain for PG hydrolysis (as illustrated in Fig. 5A for RipA). FtsEX is highly conserved in bacteria, suggesting an ancient general role in PG remodeling. In contrast, SteAB is largely restricted to Mycobacteriales . This group of bacteria has a thick waxy cell envelope formed by the plasma membrane, a two-layered cell wall composed of PG and arabinogalactan, and an unusual outer membrane composed of mycolic acids, the mycomembrane. As the cell envelope is sequentially assembled at the septal junction, the peripheral PG layer remains continuous, acting as a mechanical link that holds the daughter cells together throughout septation ( 2 ). The primary role of RipA is to cleave this stress-bearing PG layer, asymmetrically weakening the mechanical strength of the cell envelope ( 4 , 5 ) to generate a breaking point for V-snapping. This rapid, mechanically driven cell separation promoted by turgor pressure ( 2 , 46 ) is a common trait in Mycobacteriales , but rare in other bacteria ( 47 ). Accordingly, RipA deficiency results in linear chains of non-growing cells ( 4 ) that can be induced to divide by the local application of external mechanical forces ( 48 ). Understanding these specific mechanisms of cell wall remodeling and cytokinesis could offer new attractive therapeutic targets in the context of a critical human pathogen, M. tuberculosis . MATERIALS AND METHODS Bacterial strains and growth conditions Escherichia coli DH5α or CopyCutter EPI400 were used for cloning and grown in Luria-Bertani (LB) broth or agar plates at 37°C supplemented with 50 µg/mL kanamycin or 100 µg/mL carbenicillin when required. For protein production, E. coli BL21 (DE3) (for soluble proteins) and C41 ( 49 ) (for membrane proteins) cells were grown in 2YT broth supplemented with 50 µg/mL kanamycin or 100 µg/mL carbenicillin at the appropriate temperature. For the in vivo experiments, Cglu ATCC13032 was defined as the wild-type (wt) strain. All Cglu strains generated for this study ( Table S2 ) were grown at 30°C with shaking at 120 rpm in brain heart infusion (BHI) medium or CGXII minimal medium supplemented with 4% sucrose ( 50 ). When required, the BHI and CGXII media were supplemented with 25 µg/ml kanamycin (BHI kan and CGXII kan ). Knock-out strain generation of C. glutamicum CgluΔsteAB was generated using a two-step recombination strategy with the pk19mobsacB plasmid to delete the steA coding region as described previously ( 26 ). Briefly, approximately 600 bp flanking steA upstream and downstream of Cglu genomic DNA were amplified by PCR using chromosomal DNA of Cglu as a template. The PCR fragments were cloned by Gibson assembly into a linearized pk19mobsacB . The resulting plasmid was electroporated into Cglu . Successful first recombination events were confirmed by PCR and positive colonies were grown overnight in BHI kan medium. The second round of recombination was selected by growth in BHI plates containing 10% (w/v) sucrose. Kanamycin-sensitive colonies were screened by colony PCR to check for steA deletion. Positive colonies were verified by sequencing (Eurofins, France). Cloning for recombinant protein production The primers used for PCR amplification of the different fragments or site-directed mutagenesis are listed in Table S3 . Cloning was performed by assembling the purified PCR fragments into the specified pET derivative expression vector using the commercially available NEBuilder HiFi DNA Assembly Cloning Kit (New England Biolabs). For soluble protein production, M. tuberculosis steA, steB and ripA truncations were amplified by PCR using codon-optimized synthetic genes (GenScript) and cloned into a pET vector containing an N-terminal 6xHis-SUMO tag. For full-length protein production , steA and steB genes were amplified by PCR using genomic DNA of C. glutamicum as template and cloned into a pTGR5 shuttle expression vector ( 51 ), under the control of a T7 promoter and with or without an N-terminal 6xHis tag. Co-expression vectors were generated by restriction-ligation cloning. Soluble protein expression and purification All constructs were expressed in E. coli BL21 (DE3) using an autoinduction method ( 52 ). After an initial incubation of 4 h at 37°C, cells were grown for 20 h at 18°C in 2YT medium complemented with autoinduction supplement and 100 μg/mL carbenicillin. Cells were harvested, flash frozen in liquid nitrogen and stored at −20°C. Cell pellets were resuspended in lysis buffer (50 mM Hepes pH 8.0, 500 mM NaCl, 10 mM imidazole, 5% glycerol, 1 mM MgCl 2 , benzonase, lysozyme, 0.25 mM Tris (2-carboxyethyl) phosphine hydrochloride (TCEP), EDTA-free protease inhibitor cocktails (Roche) at 4°C and lysed by sonication. Cell debris were removed by centrifugation (15,000 g) for 15 min at 4°C, and the supernatant loaded onto a Ni-NTA affinity chromatography column (HisTrap FF crude, Cytiva) pre-equilibrated in buffer A (50 mM Hepes pH 8, 500 mM NaCl, 10 mM imidazole, 5% glycerol). His-tagged proteins were eluted with a linear gradient of buffer B (50 mM Hepes pH 8.0, 500 mM NaCl, 0.5 M imidazole). The fractions of interest were pooled and dialyzed in the presence of the SUMO protease at a 1:100 w/w ratio. Dialysis was carried out at 4°C overnight in SEC buffer (25 mM Hepes pH 8.0, 150 mM NaCl). For Mt SteA, a higher salt concentration (500 mM NaCl) is necessary to keep the protein soluble. Cleaved His-tags and His-tagged SUMO protease were removed with Ni-NTA agarose resin. The cleaved protein was concentrated and injected onto a Superdex 75 or 200 16/60 size exclusion column (GE Healthcare) pre-equilibrated at 4°C in SEC buffer. The peak corresponding to the protein was concentrated, flash frozen in small aliquots in liquid nitrogen, and stored at −80°C. Protein concentration was determined spectrophotometrically at 280 nm and purity was confirmed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). Se-Met-derived Cg SteA was expressed in E. coli BL21 (DE3) with all media containing 50 µg/mL carbenicillin. Cells were grown for 8 h at 37°C in 2YT medium and inoculated 1:100 in M9 medium (33.7 mM Na 2 HPO 4 -2H 2 O, 22.0 mM KH 2 PO 4 , 8.6 mM NaCl, 9.4 mM NH 4 Cl, 2 mM MgSO 4 , 0.3 mM CaCl 2 0.4% (w/v) D-glucose, 3.8 µM thiamin, 4.1 µM biotin). The overnight culture was diluted 1:50 in fresh M9 medium and grown until OD600 = 0.6. The methionine biosynthetic pathway was inhibited by adding lysine, phenylalanine, and threonine at 100 mg/L; isoleucine and valine at 50 mg/L; and selenomethionine at 60 mg/L. Protein expression was induced 30 min after addition of amino acids by adding IPTG to a final concentration of 1 mM, and cells were grown for 20 h at 18°C, harvested, and flash frozen in liquid nitrogen. Protein purification was performed as described above. Membrane protein expression and purification Cg SteA and Cg SteB-His full-length proteins were recombinantly co-expressed in E. coli C41 strain. Cells were grown in 4 L of LB media at 37°C until a OD 600 of 0.6-0.8 and then expression was induced by addition of 0.5 mM IPTG with an additional incubation of 4 hs at 30°C. Cells were harvested and flash frozen in liquid nitrogen. All following steps were performed at 4°C unless otherwise specified. Cell pellets were resuspended in Lysis buffer (50 mM Hepes pH 7.5, 300 mM NaCl, 5% glycerol, 1 mM MgCl 2 , benzonase, lysozyme, EDTA-free protease inhibitor cocktails (Roche)) and lysed through 3x passages in a CellD press (Constant Systems) at 2.1 kbar. The lysate was cleared by centrifugation (15 min, 15,000 × g ) and centrifuged in Ti45 tubes for 1 h at 100,000 x g in an Optima L-100 XP ultracentrifuge (Beckman Coulter). Pelleted membranes were resuspended with a Dounce homogenizer in Membrane buffer (50 mM Hepes pH 7.5, 500 mM NaCl, 10% glycerol, EDTA-free protease inhibitor cocktail) and solubilized upon addition of an equivalent volume of Membrane buffer containing 2.4% (w/v) DDM for 30 min at room temperature with gentle rotation. 2 mL of Ni-NTA agarose (Qiagen) slurry equilibrated in IMAC A buffer (50 mM Hepes pH = 7.5, 300 mM NaCl, 40 mM imidazole, 5% glycerol, 0.015% DDM) were added to the solubilized membrane fraction. After overnight incubation with gentle rotation, the flow-through was discarded by gravitational flow on a Poly-Prep chromatography column (BioRad), and the resin washed with 25 mL IMAC A buffer. His-tagged proteins were eluted with 5 mL of IMAC B buffer (50 mM Hepes pH = 7.5, 300 mM NaCl, 500 mM imidazole, 2.5% glycerol, 0.015% DDM). The eluate was concentrated with a Vivaspin concentrator (100 MWCO) and loaded onto a Superose 6 Increase 10/300 size exclusion (SEC) column (GE Healthcare) pre-equilibrated at 4°C in SEC buffer (25 mM Hepes pH 7.5, 150 mM NaCl) + 0.015% DDM. The elution fractions were analyzed on SDS-PAGE and the fractions of interest were pooled, concentrated, and subjected to interaction assay with Cg RipA immediately (< 24 hours at 4°C). Analytical Size Exclusion Chromatography Protein mixtures were prepared at a final concentration of 50 µM for each protein in SEC buffer, incubated at 4°C for 1 hour, centrifuged (10 mn, 15,000 × g ), and injected on a Superose 6 Increase 5/150 SEC column (GE Healthcare) pre-equilibrated with SEC buffer at 4°C. Fractions of 100 µL were collected for analysis by SDS-PAGE. SEC-SLS The oligomerization state of SteA was determined by SEC coupled to a triple detection (concentration detector: UV detector, refractometer; SLS 7°, 90°; viscometer) on a Omnisec RESOLVE and REVEAL instrument (Malvern Panalytical). SteA (100 µL sample at 1-5 mg/mL) was centrifuged for 15 min at 27,000 x g and injected on a Superdex 75 Increase 10/300 GL column (GE) pre-equilibrated in 25 mM Hepes pH 7.5, 150 mM NaCl at 20°C. External calibration was done by injecting 10 µl bovine serum albumin (BSA) at 18.3 mg/mL. The refractive index, static light scattering, and viscosity measurements were processed to determine the mass average molecular mass and the intrinsic viscosity using the OMNISEC V11.32 software (Malvern Panalytical, UK). Protein crystallization and data collection Screening for initial crystallization conditions was carried out by the sitting drop vapor diffusion method using a MosquitoTM nanoliter-dispensing system ( TTP Labtech, Melbourn, United Kingdom ) and the established protocols the Crystallography Core Facility of the Institut Pasteur ( 53 ). Promising hits were then reproduced and optimized manually using the hanging drop vapor diffusion method. All crystallization experiments were carried out at 18°C. Mt SteA (5 mg/mL), crystals grew directly in the purification buffer, 0.5 M NaCl, 25 mM Hepes pH 8, without any further manipulation. Mt SteB (11.4 mg/mL) crystals were obtained in 0.1 M CdCl 2 , 0.1 M Na Acetate pH 4.6, 30% PEG 400. Crystals of the complex between Mt SteB- Mt RipA CC were obtained in 0.01 M CoCl 2 , 0.1 M Na Acetate pH 4.6, 1 M Hexane-1,6-diol. An equimolar solution (200 μM, final concentration) of the two proteins was incubated on ice for 30 minutes prior to the crystallization experiment. Crystals of CgSteA (10 mg/mL) were grown in 0.1 M imidazole 8 pH, 0.2 M calcium acetate, 10% w/v PEG 8K, and those of SeMet-derived Cg SteA (10 mg/mL) were obtained in 0.1 M Tris pH 8, 0.2 M CaCl 2 , 0.6 M LiCl, 18% PEG 3350. Upon briefly soaking in a cryo-protectant solution containing the mother liquor supplemented by 33% (v/v) glycerol or PEG 400, crystals were flash frozen in liquid nitrogen. Diffraction data were collected at 100K at the Synchrotron facilities Soleil (Saclay, France) or ESRF (Grenoble, France). Structure determination and crystallographic refinement All diffraction data were processed using XDS ( 54 ) and Aimless from the CCP4 software suite ( 55 ) using the AutoPROC workflow ( 56 ). Crystals of the Mt SteB-RipA CC complex showed high solvent content (73%) and a strong anisotropy. Anisotropy corrections with STARANISO ( 57 ) were applied to the diffraction data from Cg SteA and Mt SteB-RipA CC crystals. Structure determination of Mt SteB was carried out using cadmium SAD phasing (cadmium was present in the crystallization solution) on a monoclinic crystal form at 2.5 Å resolution. The CRANK2 pipeline ( 58 ) within the CCP4 software suite was used to identify 24 cadmium sites and produce a model of the protein with six molecules in the asymmetric unit. This model was used as a search probe to solve the 2 Å resolution orthorhombic Mt SteB crystal form, with three independent protein molecules in the asymmetric unit. The structure of the Mt SteB-RipA CC complex was solved by molecular replacement using Mt SteB as search model. Despite strong anisotropy and high solvent content (>70%), the initial Fourier maps provided enough information to unambiguously build the missing Mt RipA CC molecule. The Mt SteA structure was solved by molecular replacement using the AlphaFold predicted coordinates of the N and C-terminal domains, separately. 20 residues corresponding to the central alpha-helix linker were removed and manually re-built in Coot to account for the relative flexibility of the two domains. The structure of Cg SteA was determined by SAD phasing at 3 Å resolution using the SeMet-labeled protein and refined against a 2 Å dataset from native Cg SteA crystals. All crystal structures underwent extensive iterative cycles of manual model building with COOT ( 59 ) and reciprocal space refinement with PHENIX ( 60 ) or BUSTER ( 61 ). Non crystallographic symmetry (when appropriate) and TLS constraints were applied during refinement. The final refinement statistics are reported in Table S1 . All molecular graphics images were generated using ChimeraX ( 62 ). AlphaFold calculations Structural predictions were performed on the High-Performance Computing (HPC) Core Facility of the Institut Pasteur computer cluster using a local installation of AlphaFold2 (v2.3.2). Model type ‘monomer’ or ‘multimer’ were used for monomeric and multimeric predictions, respectively. To ensure unbiased predictions for the different RipA forms, structural templates were disabled in these cases. All other parameters were kept as default and amber relaxation was applied to all output models. All models converged to similar conformations and only the best one for each run is shown in Figs. S4 and S10. BLI assays The affinities of purified Mt SteB and Mt RipA catalytic domain ( Mt RipA CAT , residues 261-472) towards the Mt RipA coiled coil domain ( Mt RipA CC , residues 40-240) were assessed in real-time using a bio-layer interferometry Octet-Red384 device (Pall ForteBio) at 25°C. Biotinylated Mt RipA CC was diluted at 10 μg/ mL in buffer A (25 mM HEPES pH 7.5, 150 mM NaCl, 5% glycerol) and immobilized on the commercially available Sartorius Streptavidin biosensors for 5 min at 1,000 rpm followed by a washing step in buffer A for 3 min to remove any loosely bound protein. For the biotinylation reaction, 100 μL of recombinant Mt RipA CC (25 μM) was incubated with 20 molar excess of EZ-Link NHS-PEG4-Biotin (Thermo Scientific) following supplier instructions. Empty sensors were used as reference for unspecific binding. Mt RipA CC -loaded or empty reference sensors were incubated for 5 min at 1,000 rpm in the absence and presence of serially diluted concentrations of Mt SteB (200 to 3.125 μM range) or Mt RipA CAT (80 to 1.25 μM range) in buffer A supplemented with BSA at 1 mg/mL and 0.05% Tween 20. Specific signals were obtained by double referencing, subtracting both non-specific signals measured on empty sensors and buffer signals on biotinylated Mt RipA CC -loaded sensors. Two or three independent experiments were performed for Mt SteB and Mt RipA CAT , respectively; and Kd values were obtained from steady-state signal versus concentration curves fitted with GraphPad Prism 9 assuming a one-site binding model. Nanoscale differential scanning fluorimetry (NanoDSF) assay Fluorescence measurements were carried out on the Prometheus NT.48 (NanoTemper Technologies) using standard-grade glass capillaries filled with 10–12 μl of Mt SteA or Cg SteA at 40 μM in 25 mM HEPES, pH 8, 500 mM NaCl and 5% glycerol. Prior to the experiment, protein samples were centrifuged at 15000xg for 15 minutes to remove any large aggregate. Nucleotides stock solutions were prepared in water adjusting the pH to ∼7.5 with diluted NaOH and added at a final concentration of 8 mM. An excitation power of 100% and a temperature ramp from 15 to 95°C with a slope of 1°C/min were used. The Tm was determined in the PR.ThermControl software as the maximum of the first derivative for the ratiometric (F350/F330) melting curves. All melting experiments were performed at least in triplicates. Antibiotic susceptibility assay Overnight cultures of Cglu strains were diluted to OD 600 = 0.5 in fresh BHI kan medium and serial diluted 1:10 in the same medium. 5 µL of each dilution were spotted on BHI kan plates with or without 1 µg/mL carbenicillin or 0.3 µg/mL ethambutol. Plates were imaged after 28 hours of growth at 30°C on a ChemiDoc Imaging System (Bio-Rad). Phase contrast and fluorescence microscopy For imaging, cultures were grown in BHI at 30°C for around 6 hours, pelleted at 5200 x g at RT and inoculated into CGXII, 4% sucrose and kanamycin (25 μg/mL) for overnight growth. The following day cultures were diluted to OD 600 1 in CGXII, 4% sucrose (+/− 1% gluconate) and grown for about 7 hours at 30°C to an OD 600 of about 5 (early exponential phase). For each sample, 100 μL of culture were pelleted and washed twice with fresh medium. For membrane staining, Nile Red (Enzo Life Sciences) was added to the culture (2 μg/ml final concentration) just prior to placing them on 2% agarose pads prepared with the corresponding growth medium. Cells were visualized using a Zeiss Axio Observer Z1 microscope fitted with an Orca Flash 4 V2 sCMOS camera (Hamamatsu) and a Pln-Apo 63X/1.4 oil Ph3 objective. Images were collected using Zen Blue 2.6 (Zeiss) and analyzed using the Fiji software ( 63 ), custom trained Omnipose ( 64 ) and MicrobeJ ( 65 ). Statistics and reproducibility Because of the important number of cells analyzed in each sample, Cohen’s d value was used to describe effect sizes between different strains independently of sample size: Values were interpreted as previously decribed ( 66 ), briefly the intervals of reference are considered: small (ns), d < 0.50; medium (*), 0.50 < d < 0.80; large (**), 0.80 < d <1.20; very large (***), 1.20 < d 2.0. Unless otherwise stated, p values were obtained by a Welch two sample t-test calculated on R. All experiments were performed as biological triplicates. Some autofluorescence is observed for wild- type Cglu as previously described ( 67 ). All micrographs and blots shown are representative of similar experiments carried out at least three times. Antibody production and characterization, Western Blots Polyclonal anti-SteA and anti-SteB antibodies were raised in rabbits (Covalab) against the purified soluble domains of Cg SteA and Cg SteB respectively. For antibody purification, sera from day 67 post-inoculation were purified using a 1 ml HiTrap NHS-Activated HP column (GE Healthcare) loaded with the corresponding antigen according to manufacturer instructions. Sera were diluted in binding buffer (20 mM Sodium Phosphate pH 7.4, 500 mM NaCl) and loaded onto the column, and washed with 7 ml of binding buffer. Antibodies were eluted with 10 ml elution buffer (100 mM Glycine pH 3, 500 mM NaCl) and neutralized with 1M Tris pH 9. Purified antibodies were concentrated to 8 mg/ml and mixed 1:1 with glycerol 100%, aliquoted and stored at −20°C. The characterization of the antibodies is shown in Fig. S11 ). For Western Blots, bacterial pellets of cell extracts were resuspended in lysis buffer (50 mM Bis-Tris pH 7.4; 75 mM 6-Aminocaproic Acid; 1 mM MgSO4; Benzonase and protease Inhibitor) and disrupted at 4°C with 0.1 mm glass beads and using a PRECELLYS 24 homogenizer. Total extracts (from 60 μg to 120 μg) were run on an SDS-PAGE gel, transferred onto a 0,2 μm nitrocellulose membrane and incubated for 1h with blocking buffer (5% skimmed milk, 1X TBS-Tween buffer) at room temperature (RT). Blocked membranes were incubated for 1h at RT with the corresponding primary antibody diluted to the appropriate concentration in blocking buffer. After washing in TBS-Tween buffer, membranes were probed with an anti-rabbit or an anti-mouse horseradish peroxidase-linked secondary antibody (GE healthcare) for 45 minutes. For chemiluminescence detection, membranes were washed with 1X TBS-T and revealed with HRP substrate (Immobilon Forte, Millipore). Images were acquired using the ChemiDoc MP Imaging System (Biorad). Dilutions used: anti-SteA (1:500), anti-SteB (1:1000) and anti-rabbit secondary Abs (1:10000). Data availability Atomic coordinates and structure factors have been deposited in the PDB with accession codes 9HLE ( Mt SteB), 9HMX ( Mt SteB-RipA CC ), 9HMY ( Mt SteA), 9HMZ ( CgSteA ). All materials of this paper can be provided upon reasonable request. Author contributions A.M.W. and P.M.A. designed the research; G.C., Q.G. and M.K. conducted the protein biochemistry, and purified proteins for structural and biophysical studies; Q.G. and A.S. performed molecular and cell biology and genetic experiments; Q.G. and J.P. performed cellular image analysis; G.C., Q.G. and M.M. and M.B.A carried out the biochemical and biophysical studies of protein-protein interactions; G.C., Q.G. A.H., and P.M.A. carried out the crystallographic and structure prediction studies; G.C., Q.G. and D.M. performed the sequence analyses; A.M.W. and P.M.A. wrote the paper. All authors edited the paper. Competing interests The authors declare no competing financial interests. Supplementary information Download figure Open in new tab Figure S1. Structure of Mt SteB. (A) The secondary structure topology of Mt SteB partially resembles the (Δ/α) 5 topology of receiver domains from bacterial response regulators. (B) Despite a rather low sequence identity (30%), the structural cores of Mt SteB (in blue) and Cg SteB (in orange) are closely similar. The major differences are observed in the region connecting the C-terminal globular core with the N-terminal helix, leading to a marked change in the orientation of the major helix axis, as well as at the C-terminus of the protein, which is clearly visible in Mt SteB but is absent or structurally disordered in Cg SteB. ( C ) Close up view of the dimerization interface between the helix from one protomer with the protein C-terminus from the other. Residues shown in yellow are missing in the construct of monomeric Cg SteB ( 1 ). The absence of these residues in Cg SteB, together with a weaker conservation of the heptad repeat and the lack of the TM region in the construct, might explain why Cg SteB crystallized as a monomer. Download figure Open in new tab Figure S2. Two independent BLI experiments showing the interaction profiles for the Mt RipA- Mt SteB interaction used to calculate the dissociation constant shown in Fig. 1D . Download figure Open in new tab Figure S3. (Close view of the α3-α4 loop in apo (left panel) and holo (right panel) Mt SteB, with the corresponding electron densities contoured at 1 α. Download figure Open in new tab Figure S4. The Mt RipA catalytic domain is autoinhibited by the N-terminal CC. (A) Available crystal structures of truncated forms of the Mt RipA protein (residues 265-472), showing the NlpC/P60 catalytic domain occluded by the residue segment 264-279 (PDB codes 3NE0 and 3PBC). (B) The AF-predicted models of three different Mt RipA constructs color-coded by model confidence (left panel: construct comprising residues 265-472, pLDDT=91.5; central panel: construct comprising residues 40-472, pLDDT=86.6; and right panel: construct comprising residues 40-239+265-472, pLDDT=91.4) revealed the N-terminal CC, when present, bound to the active site, as seen in the structure of Cglu RipA (PDB code 8AUC, ( 1 )). (C) Three independent BLI experiments showing the interaction profiles for the Mt RipA- Mt RipA CAT interaction used to calculate the dissociation constant shown in Fig. 1F . Download figure Open in new tab Figure S5. (A) SEC-SLS experiments show that Mt SteA elutes as a single peak with an apparent MW of 66 kDa, close to the predicted MW of the dimer (70 kDa). (B) Mt SteA secondary structure diagram. Download figure Open in new tab Figure S6. Structural similarity of the SteA N-terminal domain. ( A ) The N-terminal domain (left) can be superimposed with the phosphohistidine domains of pyruvate kinase (PDB code 2E28, ( 2 )) with a rmsd of 1.051 Å for 48 equivalent Cα positions, and the phosphoenoylpyruvate:sugar phosphotransferase system (PDB code 2HWG, ( 3 )) with a rmsd of 1.019 Å for 25 equivalent Cα positions. Structures are colored according to secondary structure ( B ) Close view of superposed SteA (green), 2E28 (tan) and 2HWG (pink). The (phospho)histidine residue is missing in Mt SteA. The partial sequence alignment of this region is shown at right. Download figure Open in new tab Figure S7. Structural flexibility of the SteA N-terminal domain. (A) Superposition of the 8 crystallographically independent molecules of Cg SteA color-coded according to B values. The highest B values are found for the N-terminal domain, on the right. (B) Two different views of the superposition between Mt SteA (green) and Cg SteA (orange, one molecule). Download figure Open in new tab Figure S8. The phosphonucleotide-binding pocket of SteA. ( A ) The conserved binding pockets of Mt SteA (top) and Cg SteA (bottom) are both predicted to bind carbohydrate using PeSTo-Carbs, a Deep-Learning approach trained on protein-carbohydrate interfaces ( 4 ). ( B ) Binding of different phosphonucleotides to Mt SteA as assessed by nanoDSF were inconclusive. However, the same experiment carried out with Cg SteA (see Fig.3C , Main Text) indicated that the protein would bind UDP- or GDP-containing compounds. Download figure Open in new tab Figure S9. (A) Transcription start sites as described in ( 5 ) (B) Western Blots anti-SteA (top) and anti-SteB (bottom) of whole cell extracts (60 μg) from the different strains shown in Fig. 4 . An arrow indicates the specific signal for Cg SteA and Cg SteB, respectively. Download figure Open in new tab Figure S10. AlphaFold-predicted model of the heterotetramer ( Mt SteA/ Mt SteB) 2 color-coded according to model confidence (left panel, plDDT=84) or protein organization (right panel), with the Mt SteA dimer shown in green and the Mt SteB dimer in blue. Download figure Open in new tab Figure S11. Antibody characterization. ( A ) Western Blot using purified anti-SteA antibody. Lane 1: molecular weight markers [kDa]; lane 2: total cell extract (120 μg) of Cglu ; lane 3: total cell extract (120 μg) of Cglu_λ1steAB ; lane 4: recombinant full-length His-SteA (0.1 μg); lane 5: recombinant soluble His-SteA (0.4 μg) ( B ) Western Blot using purified anti-SteB antibody. Lane 1: molecular weight markers, kDa; lane 2: total cell extract (120 μg) of Cglu ; lane 3: total cell extract (120 μg) of Cglu_empty plasmid ; lane 4: total cell extract (120 μg) of Cglu_λ1steAB ; lane 5: total cell extract (120 μg) of Cglu_λ1steAB + Scarlet-SteB (note that in these conditions Scarlet-SteB is not expressed); lane 6: recombinant soluble His-SteB (8 ng). View this table: View inline View popup Download powerpoint Supplementary Table S1. Crystallographic data collection and refinement statistics. View this table: View inline View popup Download powerpoint Supplementary Table S2. Plasmids and strains used in this study. View this table: View inline View popup Download powerpoint Supplementary Table S3. Oligonucleotide primers used in this study. Acknowledgements We gratefully acknowledge the core facilities at the Institut Pasteur C2RT, P. England, B. Raynal, S. Brûlé (PFBMI); P. Weber, C. Pissis, A. Mechaly (PFC), J. Fernandes (UtechS PBI / Imagopole, supported by France BioImaging; ANR-10–INSB–04; Investments for the Future) and the HPC Core Facility of the Institut Pasteur. We also acknowledge the synchrotron sources Soleil (Saint-Aubin, France) and ESRF (Grenoble, France) for granting access to the facilities, and the staff of Proxima 1, Proxima 2A and ID-23-1 beamlines for helpful assistance during X-ray data collection. This work was partially supported by grants from the Agence Nationale de la Recherche (ANR, France), contracts ANR-18-CE11-0017 (P.M.A.), ANR-21-CE11-0003 (A.M.W.), and ANR-21-CE20-0040 (A.M.W), from the Fondation pour la Recherche Médicale (grant number EQU202303016284 to P.M.A.) and by institutional grants from the Institut Pasteur, the CNRS, and Université Paris Cité. For the purpose of open access, the author has applied a CC-BY public copyright licence to any Author Manuscript version arising from this submission. 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