Characterization of the Pilotin-Secretin Complex from the Salmonella enterica Type III Secretion System Using Hybrid Structural Methods

•The pilotin InvH forms an ?-helical homodimer when crystallized and in solution•The secretin InvG binds a 1:1 ratio along hydrophobic groove•InvH homodimerizes using the same interface The type III secretion system (T3SS) is multi-membrane-spanning protein channel used by Gram-negative pathogenic bacteria to secrete effectors directly into host cell cytoplasm. In many species reliant on T3SS for pathogenicity, proper assembly of outer membrane pore depends diverse family lipoproteins called pilotins. We present structural biochemical data Salmonella enterica S domain its cognate InvG. Characterization X-ray crystallography revealed dimerized, pilotin. Size-exclusion-coupled multi-angle light scattering small-angle provide supporting evidence formation solution. Structures InvH-InvG heterodimeric complex determined NMR spectroscopy indicate predominantly interface. Knowledge interaction between brings us closer understanding mechanisms which pilotins assemble pore. pathogenicity clinically relevant relies virulence nanomachine termed (T3SS), or injectisome (Deng et al., 2017Deng W. Marshall N.C. Rowland J.L. McCoy J.M. Worrall L.J. Santos A.S. Strynadka N.C.J. Finlay B.B. Assembly, structure, function regulation systems.Nat. Rev. Microbiol. 2017; 15: 323-337Crossref PubMed Scopus (256) Google Scholar). manner syringe, this proteinaceous allows passage effector proteins from bacterial cytosol through hollow needle filament pore-forming tip infected host. secreted serve target manipulate processes, resulting various effects. As such, essential important clinical community pathogens, including enterica, Pseudomonas aeruginosa, Bordetella pertussis. Whereas that are translocated vary greatly nature based each species' life cycle niche, apparatus itself well conserved. salmonellosis, infection gut non-typhoidal S. invasion cells carried out encoded island 1 (SPI-1). discovery novel antibiotic targets lags severely behind rise antibiotic-resistant other Enterobacteriaceae (Wang 2019Wang X. Biswas Paudyal N. Pan H. Li Fang Yue M. Antibiotic resistance salmonella typhimurium isolates recovered food chain national antimicrobial monitoring 1996 2016.Front. 2019; 10: 985Crossref (71) Scholar), mechanics vital it development new anti-virulence therapeutics. ?3 MDa composed over 20 different proteins, creating concentric rings repeating units pass inner membranes It has three main subcomplexes: cytosolic sorting platform, with roles substrate selection energizing (Lara-Tejero, 2019Lara-Tejero Type Secretion System Sorting Platform. Springer Berlin Heidelberg, 2019Crossref (5) Scholar; Minamino 2011Minamino T. Morimoto Y.V. Hara Namba K. An energy transduction mechanism flagellar export.Nat. Commun. 2011; 2: 475Crossref (98) Scholar, 2016Minamino Aldridge P.D. export gate dual fuel engine can use both H+ Na+ export.PLOS Pathog. 2016; 12: e1005495Crossref (43) Scholar); basal body, passes (IM), peptidoglycan wall, (OM) (Worrall 2016Worrall Hong C. Vuckovic Deng Bergeron J.R.C. Majewski D.D. Huang R.K. Spreter Yu Z. al.Near-atomic-resolution cryo-EM analysis T3S body.Nature. 540: 597-601Crossref (78) translocon pore, create (Dey 2019Dey Chakravarty A. Guha P. De Guzman R.N. needle, tip, translocon.Protein Sci. 28: 1582-1593PubMed Assembly such no mean feat, ever-evolving models have been proposed explain observations collected prototypical T3SS, as range (recently reviewed Experiments ongoing test these more accurately describe modes individual components T3SS. OM body occurs highly regulated that, although generally conserved, differs details targeting steps (Howard 2019aHoward S.P. Estrozi L. Bertrand Q. Contreras-Martel Strozen Job V. Martins Fenel D. Schoehn G. Dessen Pilotin Vibrio vulnificus 2 system, EpsS..10.2210/pdb6I2V/pdbDate: 2019Google single multi-domain polypeptide oligomerizes at massive (?1 MDa) gated, double-walled ? barrel 15 subunits 60 strands (Hay 2017aHay I.D. Belousoff M.J. Lithgow Structural basis engagement membranes.MBio. 8 (e01344-17)Crossref (27) Yan 2017Yan Yin Xu Zhu Y. insights translocation II system.Nat. Struct. Mol. Biol. 24: 177-183Crossref (65) 2018Yin insight pilotin–secretin enterotoxigenic Escherichia coli.Nat. 2018; 3: 581-587Crossref (17) member broadly conserved significant sequence conservation among distinct systems, (T2SS), IV pilus (T4PS), filamentous phage extrusion (Majewski 2018Majewski Secretins revealed: giant gated portals bacteria.Curr. Opin. 51: 61-72Crossref (13) Secretin monomers transported periplasm general Sec upon oligomerization undergo BAM-independent insertion (Dunstan 2015Dunstan R.A. Hay Wilksch J.J. Schittenhelm R.B. Purcell A.W. Clark J. Costin Ramm Strugnell pores GspD, Wza CsgG does not require Omp85 BamA TamA: pores.Mol. 2015; 97: 616-629Crossref (35) most cases, process mediated T3SS-specific chaperone known Pilotins small anchored leaflet via localization (Lol) pathway; they localize while protecting degradation (Collin 2011Collin Guilvout I. Nickerson N.N. Pugsley A.P. integral lipoprotein-specific Lol pathway dedicated lipoprotein pilotin: protein.Mol. 80: 655-665Crossref (47) Koo 2012Koo Burrows L.L. Lynne Howell Decoding accessory escort services.FEMS Lett. 2012; 328: 1-12Crossref (45) T2SS bind ?50 residue C-terminal region their secretins. These regions form helix-loop-helix structure (Daefler 1997Daefler Hardie K.R. Russel PulD contains binding site chaperone, PulS, confers PulS dependence pIVf1 function.Mol. 1997; 465-475Crossref (84) knockouts disrupt subsequent effects, mis-targeting IM (Salmonella) entirely preventing (Crago Koronakis, 1998Crago A.M. Koronakis ring-like multimer requires localization.Mol. 1998; 30: 47-56Crossref (135) Daefler Russel, 1998Daefler required InvG.Mol. 1367-1380Crossref (90) Dunstan 2013Dunstan Heinz E. Wijeyewickrema L.C. Pike Evans T.J. Praszkier Robins-Browne R.M. Korotkov K.V. al.Assembly found vibrio cholerae AspS.PLoS 2013; 9: e1003117Crossref (48) Despite similar functions, proteins. Most share little identity, differing predicted secondary structures. Of pilotins, two crystallographic structures ?-sheet-containing proteins: aeruginosa ExsB ?-sandwich fold (Izoré 2011Izoré Perdu Attree Faudry characterization exsb aeruginosa.J. 413: 236-246Crossref (21) whereas Shigella flexneri MxiM cracked ?-barrel deep cavity capable either lipids (Lario 2005Lario P.I. Pfuetzner Frey E.A. Creagh Haynes Maurelli A.T. Structure pilot protein.EMBO 2005; 1111-1121Crossref (61) Okon 2008Okon Moraes T.F. Lario A.L. C.A. McIntosh L.P. type-III pilot-secretin flexneri.Structure. 2008; 16: 1544-1554Abstract Full Text PDF GspS, OutS, are, hand, compact made up four ? helices (Gu 2012Gu Rehman Wang Shevchik V.E. Pickersgill R.W. functional pilotin-secretin system.PLoS 8: e1002531Crossref (37) Hol, 2013Korotkov Hol W.G.J. Crystal enterohemorrhagic coli system.J. 182: 186-191Crossref (9) 2013Rehman Gu Anatomy Dickeya dadantii pilotin.Acta Crystallogr. Section D 69: 1381-1386Crossref Tosi 2011Tosi Mollica Jensen M.R. Blackledge Baron B. England Pilotin-secretin recognition Klebsiella oxytoca: interactions T2SS.Mol. 82: 1422-1432Crossref (31) AspS EspS contain mixture sheets Howard T4PS still, forming much larger bundles tetratricopeptide repeats lacking identified (Kim 2006Kim Oh Han Kim E.E. Lee PilF: implication 4 biogenesis aeruginosa.Biochem. Biophysical Res. 2006; 340: 1028-1038Crossref (46) 2008Koo Tammam Ku S.-Y. Sampaleanu L.M. P.L. PilF multimerization secretin.J. Bacteriol. 190: 6961-6969Crossref (75) Trindade 2008Trindade M.B. Pelicic widely factor affects stability multimers.J. 378: 1031-1039Crossref (38) complexes MxiM, AspS, respective domains (MxiD, OutX, AspX), characterized atomic level architecture, all corresponding lined aromatic residues. Historically, SPI-1 best-studied However, had structurally prior study. Predicted be primarily helical, shares identity targeting, but oligomerization, Knockout invH results IM, reducing efficiency 70%–80% C57BL/6 mice (Pati 2013Pati N.B. Vishwakarma Jaiswal Periaswamy Hardt W.-D. Suar Deletion gene serovar Typhimurium limits Sip proteins.Microbes Infect. 66-73Crossref (22) player and, given periplasmic localization, relatively accessible unexplored target. Here, we InvH, apo bound domain, spectroscopy. Along complementary experiments, show absence InvG, heterodimer complexed domain. suggests alternative those previously monomeric ?-barrel-fold (Okon To gain better broad diversity structures, sought crystallize heterologously expressed InvH. Secondary Phyre2 joined long (?50 residue) disordered linker signature (L-A/S-G/A-C) N-terminal lipobox (Kelley 2015Kelley L.A. Mezulis Yates C.M. Wass M.N. Sternberg M.J.E. web portal modeling, prediction analysis.Nat. Protoc. 845-858Crossref (5007) Guided predictions, focused crystallization trials high-yielding soluble constructs: InvH27-147, sequence, InvH70-147, truncated past (Figure 1A). InvH27-147 was failed crystallize, likely due region. microbatch approach under oil, InvH70-147 formed large plate-like crystals. Single-wavelength anomalous diffraction phasing mercury acetate-soaked crystals successful, final native dataset refined 1.15 Å resolution 1B, Table 1).Table 1X-Ray Crystallography Data Collection, Refinement, Validation StatisticsInvH70-147InvH70-147 Hg2+ SoakedInvH84-147/InvG543-558Data CollectionWavelength (Å)0.981.011.00Resolution (Å)44.33–1.20 (1.24–1.20)44.28–1.37 (1.42–1.37)31.45–1.85 (1.92–1.85)Space groupP 1P 65 2Unit parameters (Å)a = 25.5, b 53.2, c 56.9? 107.7°, 97.2°, ? 96.0°a 25.4, 107.4°, 96.1°a 36.3, 221.6? 90°, 120°Total reflections325,986418,857135,602Unique reflections82,54036,4398,226Multiplicity3.9 (3.9)11.5 (9.4)16.5 (17.1)Completeness (%)94.5 (91.2)61.6 (4.7)99.5 (99.8)Mean I/?I11.1 (2.0)27.7 (1.8)16.8 (2.5)Wilson B (Å2)7.712.141.4Rmerge0.083 (0.765)0.061 (0.948)0.069 (0.885)Rmeas0.097 (0.886)0.063 (1.002)0.071 (0.912)Rpim0.049 (0.446)0.019 (0.315)0.018 (0.218)CC1/20.998 (0.681)0.999 (0.713)1.000 (0.908)CC?0.999 (0.900)1.000 (0.912)1.000 (0.976)RefinementReflections refinement82,5408,225Reflections Rfree4,111420Rwork/Rfree13.2/16.821.8/24.7CCwork/CCfree (%)96.8/95.490.6/91.4Number non-hydrogen atoms3,048708 Macromolecules2,605629 Ligands3810 Solvent40569Protein residues30674RMS (bonds, Å2)0.0160.019RMS (angles,°)1.861.88Ramachandran favored (%)99.7100.0Ramachandran allowed (%)0.30.0Ramachandran outliers (%)0.00.0Rotamer (%)0.30.0Clashscore1.90.8Average (Å2)17.048.3 Macromolecules14.846.3 Ligands21.059.8 Solvent31.064.9Number TLS groupsNone2 Open table tab asymmetric unit consists molecules S1A), homodimers (Figures 1C 1D). No differences backbone architecture were noted dimers, pairwise C? root-mean-square deviations (RMSDs) ?0.2 Å. compact, per subunit. Helix kinked, halves ?1a ?1b. (residues 73–88) interacts solely opposing protomer, burying surface Phe99, Phe119, Leu123, Ile133, Leu137 1A 1E). Following turn after ?1a, ?1b (90–101) ?2 (111–124) wide hairpin. 10-residue also 4-residue helix. A loop separates ?3 (133–146), ?1b, ?2, protomer's resembles swap protomers; however, short length unlikely could addition described above, ?1,200 Å2 dimer (calculated PDBePISA; Krissinel Henrick, 2007Krissinel Henrick Inference macromolecular assemblies crystalline state.J. 2007; 372: 774-797Crossref (6151) Scholar) involves polar loops connecting S1B). Several electron-dense observed protein-protein contacts crystal. initially densities cadmium ions, solution included 80 mM chloride (CdCl2). Creating difference map several high-intensity peaks (>5 ?), bordered non-anomalous S1C). Based pattern, scatterers cadmiums, neighbored chlorides. Although groups throughout unit, notable cluster bridging dimerization cadmiums coordinated Cys85 contact Lys89, Thr91, Lys92 1F). One localized kink coordination Gln90 Glu93 S1D). Asp111 Glu113 bridged cadmium. determine whether purified ions discussed sites, performed inductively coupled plasma mass spectrometry samples. metal elements tested (cadmium, copper, manganese, nickel, lead, zinc; shown). Therefore, CdCl2 aid crystal formation, represent physiologically species. shown Thus, size-exclusion-coupled (SEC-MALS) experiments investigate states molecular masses constructs approximately corresponded homodimer, additional monomer population 2A). further characterize solution, in-house (SAXS) S2A–S2C). Consistent SEC-MALS result, SAXS resulted calculated indicative (Table S1). fit obtain consistent envelopes averages construct generated 10 2B). case size shape envelope matched dimer, slightly wider flatter Using software Crysol generate simulated curve poor experimental (?2 17.5; attribute dynamic motion below; Figure S2D). construct, includes region, fitting produced density extending poles correspond locations N termini where residues would joined. Collectively, confirm additionally position mutated probe reflects conformation mutants within context construct. Phe84 Ile133 individually alanine opposite protomer. addition, chosen because 2C). Each Superdex 75 size-exclusion column predominant elution peak assayed SEC-MALS. I133A mutant strongest effect, shifting state 2D S3A). F84A mutation deleterious dimerization, lesser extent, dimeric detected. Both minor high-molecular-mass populations, attributed aggregation. C85A eluted peak, confirming self-association outside condition Unexpectedly, investigated series InvH84-147, still despite truncation protomer-bridging helix ?1a. suggest 70–83 role formation. importance hypothesized lie ability guide state, premature IM. separated complex. N-terminally His-tagged InvG520-562 cryoelectron microscopy (cryo-EM) assembled This fragment encompasses entirety pilotin-binding S-domain motif. high affinity body.N