X-ray crystal structure of the N-terminal domain of Staphylococcus aureus cell-cycle protein GpsB

X-ray crystal structure of the N-terminal domain of Staphylococcus aureus cell-cycle protein GpsB | 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 Confirmatory Results X-ray crystal structure of the N-terminal domain of Staphylococcus aureus cell-cycle protein GpsB View ORCID Profile Nathan I. Nicely , View ORCID Profile Thomas. M. Bartlett , View ORCID Profile Richard W. Baker doi: https://doi.org/10.1101/2025.08.05.668015 Nathan I. Nicely 1 Department of Pharmacology, UNC Chapel Hill School of Medicine ; Chapel Hill, NC 27599, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Nathan I. Nicely Thomas. M. Bartlett 2 Division of Genetics. Wadsworth Center, New York State Department of Health ; Albany, NY 12208, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Thomas. M. Bartlett For correspondence: Thomas.Bartlett{at}health.ny.gov baker{at}med.unc.edu Richard W. Baker 3 Department of Biochemistry and Biophysics, UNC Chapel Hill School of Medicine ; Chapel Hill, NC 27599, USA 4 UNC Lineberger Comprehensive Cancer Center. UNC Chapel Hill School of Medicine ; Chapel Hill, NC 27599, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Richard W. Baker For correspondence: Thomas.Bartlett{at}health.ny.gov baker{at}med.unc.edu Abstract Full Text Info/History Metrics Preview PDF Abstract GpsB is a conserved cell-cycle regulator in the Firmicute clade of Gram-positive bacteria that coordinates multiple aspects of envelope biogenesis. Recent studies demonstrate interactions between GpsB and the key division cytoskeleton FtsZ, suggesting that GpsB links cell division to various aspects of cell envelope biogenesis in Staphylococcus aureus and potentially other Firmicutes. We determined a 1.7 Å resolution crystal structure of the N-terminal domain of Staphylococcus aureus GpsB, revealing an asymmetric dimer with a bent conformation. This conformation is nearly identical to one of two conformations reported by Sacco, et al., confirming the unique conformation of S. aureus GpsB compared to other gram-positive bacteria. This structural agreement provides strong validation of the S. aureus GpsB fold and supports its proposed role in organizing the cell division machinery. Description GpsB is a widely conserved adaptor protein in Gram-positive Firmicutes (synonym Bacillota) that coordinates cell-cycle progression by coupling cell envelope biogenesis to the cell-division machinery. GpsB has two structured domains — an N-terminal dimerization domain and a C-terminal trimerization domain — which are separated by a flexible linker 1 , 2 . Thus, GpsB is thought to act as a hexamer that can bind to multiple cell division proteins simultaneously, thereby facilitating the spatiotemporal coordination of various molecular machines. The N-terminal domain of GpsB, in addition to containing a membrane-binding loop that drives binding to the inner leaflet of the membrane 1 , also binds small peptides bearing a consensus (S/T)-R-X-X-R−(R/K) motif. This motif is found in proteins such as PBP1A 3 , FtsZ 4 , 5 , EzrA 6 , DivIVA 7 , TarO/G 5 , and FacZ 8 , allowing GpsB to coordinate division site localization of multiple protein complexes. Consistent with these binding motifs, GpsB directly binds and regulates FtsZ 4 , contributing to the correct placement of FtsZ and other Staphylococcal division proteins 8 and coordinating cell envelope growth to division 5 , underlining its intriguing potential to communicate information between cell envelope synthesis and morphogenetic factors. Unsurprisingly, and presumably as a result of its coordination of these features, GpsB is necessary for normal S. aureus morphogenesis 9 , 10 . GpsB’s interactions with FtsZ 11 and various cell envelope synthesis factors 3 are conserved in other Firmicutes, suggesting its role as an adaptor between cell cycle and envelope growth is also broadly conserved. Prior to 2024, structures for the N-terminal domain of GpsB had been described for Bacillus subtilis 3 , 12 , Listeria monocytogenes 3 , and Streptococcus pneumoniae 3 . These GpsB orthologs adopt a conserved fold, showing a long parallel two-helix bundle with two short helices that form a 4-helix ‘cap’ at one end 1 . Notably, the central helical bundle is a rigid helix, reinforcing the model of GpsB as a linear adaptor scaffold. Recently, Sacco et al. revealed a novel conformation of S. aureus GpsB ( Sa GpsB) 13 , where the N-terminal homodimer adopts an asymmetric dimer, in which two protomers display a kinked helix conformation, mediated by a hinge formed by a three-residue insertion exclusive to Staphylococcus species. This hinge comprises a cluster of methionine residues (“MAD” or “MNN” insertion) not found in other Firmicutes, conferring conformational flexibility. Excising this insertion increases thermal stability and abolishes an overexpression lethal phenotype in Bacillus , suggesting functional tuning via flexibility. Thus, functional and structural divergence appears between S. aureus and other Gram-positives. Whereas GpsB in other species is rigid, Sa GpsB seems conformationally dynamic, possibly acting as a regulatory switch in divisome assembly. We independently crystallized the N-terminal domain (residues 1–75) of Sa GpsB and determined its structure at 1.7 Å resolution ( Figure 1A , Table 1 ). Our analysis shows an asymmetric dimer with a kinked helix conformation, in excellent agreement with the GpsB dimers seen in 8E2B.pdb (chains C/D) and 8E2C.pdb (chains A/B). Root-mean-square deviation (RMSD) between our model and 8E2B chains C/D is ~0.4 Å over all Cα atoms (70 residues), underscoring the similarity of the two structures. While 8E2B contains two Sa GpsB dimers in the asymmetric unit, a dimer with a kinked helix of approximately 20 ° and a dimer with a kinked helix of approximately 40 ° , our structure best matches the 40 ° bent helix conformation ( Figure 1A ). Both described Sa GpsB conformations vary significantly from the nearly straight helices observed in Bs GpsB, Lm GpsB, and Sp GpsB. View this table: View inline View popup Download powerpoint Table 1. X-ray collection and refinement statistics Download figure Open in new tab Figure 1. X-ray structure of Sa GpsB residues 1-75 A. Comparison of Bs GpsB (yellow), Sa GpsB conformation 1 (pink), Sa GpsB conformation 2 (green), and Sa GpsB from this study (blue). Each GpsB model is colored with two different shades of the same color to show the dimeric assembly.an overlay of the 40 kinked helix conformation from 8E2B and the 40 kinked helix structure from this study is shown (far right). B. Crystal packing for our Sa GpsB crystal structure (left) is compared with crystal packing for Sa GpsB + FtsZ peptide (8E3C.pdb; right). 8E2B has two copies of the GpsB dimer and was therefore not shown. While our crystal structure and 8E2C both have a single copy of GspB dimer in the asymmetric unit, their crystal packing arrangement is unique, demonstrating that the conformation observed is not influenced by the crystal lattics. C. Zoom-in of the membrane binding loop (aa 17-27) of the Sa GpsB dimer. The two conformations observed have loop displacements of 2.5-3.0 Å (yellow dashes). Importantly, our analysis shows a unique crystal packing morphology compared to 8E2B and 8E2C ( Figure 1B ), confirming that the kinked-helix conformation is not an artifact of specific crystal-packing conditions. Our data therefore supports the model proposed by Sacco et al., whereby the hinge-mediated flexibility serves as a dynamic switch in S. aureus . Although no ligand was present in our crystals, the conformation observed is virtually identical to Sa GpsB bound to the (S/T)-R-X-X-R−(R/K) motif of PBP4 (8E2C.pdb), suggesting the asymmetric dimer is intrinsic to Sa GpsB’s fold, not induced by ligand binding. Thus, this conformation likely represents the physiologically relevant state that mediates binding to partners. The only significant difference between our structural analysis is slight heterogeneity in the membrane-bending loop (residues ~17–27) ( Figure 1C ), which may reflect dynamic motion of the hinge region, as suggested in Sacco, et al. This heterogeneity is expected for a loop region proposed to insert into the inner leaflet of the membrane 1 . Our independent crystal structure of the Sa GpsB N-terminal domain validates and reinforces the asymmetric, hinge-mediated conformation first described by Sacco et al. These findings support the hypothesis that hinge flexibility enables regulatory control of divisome component assembly by modulating GpsB interactions with other binding partners. Together, this work affirms GpsB’s role as a dynamic adaptor in S. aureus cell-division and provides a solid structural foundation for further functional studies. Methods GpsB 1-75 purification GpsB residues 1-75 (from S. aureus strain NCTC 8325; Uniprot Q2FYI5; SAOUHSC_01462) were recombinantly purified as a fusion with an N-terminal 10xHis-SUMO tag. Briefly, BL21 (DE3) E. coli transformed with the expression plasmid were grown in Lysogeny Broth (LB) with 50 μg/mL kanamycin to mid log phase (OD 600 ~0.6) and induced with 0.5 mM Isopropyl β-D-1-thiogalactopyranoside (IPTG) at 18 C overnight. Cells were harvested in lysis buffer (20 mM HEPES, pH 7.5; 500 mM NaCl, 20 mM Imidazole, 1 mM DTT, 1 mM PMSF), lysed by sonication, and clarified via centrifugation at 28,000 g . Lysate was applied to Ni+-NTA resin (GoldBio), washed with high salt buffer (20 mM HEPES, pH 7.5, 1000 mM NaCl, 20 mM Imidazole), low salt buffer (20 mM HEPES, pH 7.5, 100 mM NaCl, 20 mM Imidazole), and eluted with elution buffer (20 mM HEPES, pH 7.5, 100 mM NaCl, 300 mM Imidazole). Protein was dialyzed overnight into crystallization buffer (20 mM HEPES, pH 7.5, 100 mM NaCl, 1 mM DTT), along with SUMO protease to cleave the purification tag. The following day, the uncleaved protein and free His-SUMO tag were removed by passing the eluent over Ni+-NTA resin. The protein was further purified by anion exchange and size exclusion chromatography. The protein was concentrated to 5 mg/mL. GpsB 1-75 crystallization, data collection, and model building Protein was tested for crystallization against common commercially available crystal screens using a Mosquito dropsetter (SPT Labtech) with drops composed of 200 nl protein and 200 nl reservoir solution set over 30 μl reservoir volumes. Crystals were observed within one week over a reservoir solution composed of 1.0 M Succinic Acid, 0.1 M HEPES pH 7.5, 1 %w/v PEG 2000 MME. The crystals were briefly soaked in reservoir supplemented with 15% ethylene glycol then cryocooled in liquid nitrogen. Diffraction data were collected at Southeast Regional Collaborative Access Team (SER-CAT) 22-ID beamline at the Advanced Photon Source, Argonne National Laboratory, using an incident beam of 1 Å in wavelength. Data were reduced in HKL-2000 14 . The structure was phased by molecular replacement using Phaser 15 with PDB 8e8b as the search model 13 . Real space rebuilding were done in Coot 16 , and reciprocal space refinements and validations were done in PHENIX 17 . Coordinates and structure factors have been deposited in the Protein Data Bank (PDB) with accession number 9PV2. Acknowledgements This research used resources of the Advanced Photon Source, a U.S. Department of Energy (DOE) Office of Science user facility operated for the DOE Office of Science by Argonne National Laboratory under Contract No. DE-AC02-06CH11357. SER-CAT is supported by its member institutions, equipment grants (S10_RR25528, S10_RR028976 and S10_OD027000) from the National Institutes of Health, and funding from the Georgia Research Alliance. References 1. ↵ Halbedel , S. & Lewis , R. J. Structural basis for interaction of DivIVA/GpsB proteins with their ligands . Mol. Microbiol . 111 , 1404 – 1415 ( 2019 ). OpenUrl CrossRef PubMed 2. ↵ Hammond , L. R. , White , M. L. & Eswara , P. J. vIVA la DivIVA! J . Bacteriol . 201 , e00245 – 19 ( 2019 ). OpenUrl PubMed 3. ↵ Cleverley , R. M. et al. The cell cycle regulator GpsB functions as cytosolic adaptor for multiple cell wall enzymes . Nat. Commun . 10 , 261 ( 2019 ). OpenUrl CrossRef PubMed 4. ↵ Eswara , P. J. et al. An essential Staphylococcus aureus cell division protein directly regulates FtsZ dynamics . eLife 7 , e38856 ( 2018 ). OpenUrl CrossRef PubMed 5. ↵ Hammond , L. R. et al. GpsB Coordinates Cell Division and Cell Surface Decoration by Wall Teichoic Acids in Staphylococcus aureus . Microbiol. Spectr . 10 , e0141322 ( 2022 ). OpenUrl 6. ↵ Steele , V. R. , Bottomley , A. L. , Garcia-Lara , J. , Kasturiarachchi , J. & Foster , S. J. Multiple essential roles for EzrA in cell division of Staphylococcus aureus . Mol. Microbiol . 80 , 542 – 555 ( 2011 ). OpenUrl CrossRef PubMed 7. ↵ Bottomley , A. L. et al. Coordination of Chromosome Segregation and Cell Division in Staphylococcus aureus . Front. Microbiol . 8 , 1575 ( 2017 ). OpenUrl CrossRef PubMed 8. ↵ Bartlett , T. M. et al. FacZ is a GpsB-interacting protein that prevents aberrant division-site placement in Staphylococcus aureus . Nat. Microbiol . 9 , 801 – 813 ( 2024 ). OpenUrl PubMed 9. ↵ Sutton , J. A. F. et al. The roles of GpsB and DivIVA in Staphylococcus aureus growth and division . Front. Microbiol . 14 , ( 2023 ). 10. ↵ Costa , S. F. et al. The role of GpsB in Staphylococcus aureus cell morphogenesis . mBio 15 , e03235 – 23 ( 2024 ). OpenUrl PubMed 11. ↵ Bhattacharya , D. , King , A. , McKnight , L. , Horigian , P. & Eswara , P. J. GpsB interacts with FtsZ in multiple species and may serve as an accessory Z-ring anchor . Mol. Biol. Cell 36 , ar10 ( 2025 ). OpenUrl PubMed 12. ↵ Rismondo , J. et al. Structure of the bacterial cell division determinant GpsB and its interaction with penicillin-binding proteins . Mol. Microbiol . 99 , 978 – 998 ( 2016 ). OpenUrl CrossRef PubMed 13. ↵ Sacco , M. D. et al. Staphylococcus aureus FtsZ and PBP4 bind to the conformationally dynamic N-terminal domain of GpsB . eLife 13 , e85579 ( 2024 ). OpenUrl PubMed 14. ↵ Otwinowski , Z. & Minor , W. Processing of X-ray diffraction data collected in oscillation mode . Methods Enzymol . 276 , 307 – 326 ( 1997 ). OpenUrl CrossRef PubMed 15. ↵ McCoy , A. J. et al. Phaser crystallographic software . J. Appl. Crystallogr . 40 , 658 – 674 ( 2007 ). OpenUrl CrossRef PubMed Web of Science 16. ↵ Emsley , P. , Lohkamp , B. , Scott , W. G. & Cowtan , K. Features and development of Coot . Acta Crystallogr. D Biol. Crystallogr . 66 , 486 – 501 ( 2010 ). OpenUrl CrossRef PubMed Web of Science 17. ↵ Liebschner , D. et al. Macromolecular structure determination using X-rays, neutrons and electrons: recent developments in Phenix . Acta Crystallogr. Sect. Struct. Biol . 75 , 861 – 877 ( 2019 ). 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