Structural and Functional Insights into the Pilotin-Secretin Complex of the Type II Secretion System

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Gram-negative bacteria secrete virulence factors and assemble fibre structures on their cell surface using specialized secretion systems. Three of these, T2SS, T3SS and T4PS, are characterized by large outer membrane channels formed by proteins called secretins. Usually, a cognate lipoprotein pilot is essential for the assembly of the secretin in the outer membrane. The structures of the pilotins of the T3SS and T4PS have been described. However in the T2SS, the molecular mechanism of this process is poorly understood and its structural basis is unknown. Here we report the crystal structure of the pilotin of the T2SS that comprises an arrangement of four α-helices profoundly different from previously solved pilotins from the T3SS and T4P and known four α-helix bundles. The architecture can be described as the insertion of one α-helical hairpin into a second open α-helical hairpin with bent final helix. NMR, CD and fluorescence spectroscopy show that the pilotin binds tightly to 18 residues close to the C-terminus of the secretin. These residues, unstructured before binding to the pilotin, become helical on binding. Data collected from crystals of the complex suggests how the secretin peptide binds to the pilotin and further experiments confirm the importance of these C-terminal residues in vivo.

Vyšlo v časopise: Structural and Functional Insights into the Pilotin-Secretin Complex of the Type II Secretion System. PLoS Pathog 8(2): e32767. doi:10.1371/journal.ppat.1002531
Kategorie: Research Article
prolekare.web.journal.doi_sk: https://doi.org/10.1371/journal.ppat.1002531

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1. BayanNGuilvoutIPugsleyAP 2006 Secretins take shape. Mol Micro 60 1 4

2. CollinsRFFryeSAKitmittoAFordRCTonjumT 2004 Structure of the Neisseria meningitidis outer membrane PilQ secretin complex at 12 angstrom resolution. J Biol Chem 279 39750 39756

3. ReichowSLKorotkovKVHolWGJGonenT 2010 Structure of the cholera toxin secretion channel in its closed state. Nature Struct Mol Biol 17 1226 1233

4. ChamiMGuilvoutIGregoriniMRemigyHWMullerSA 2005 Structural insights into the secretin PulD and its trypsin-resistant core. J Biol Chem 280 37732 37741

5. HodgkinsonJLHorsleyAStabatDSimonMJohnsonS 2009 Three-dimensional reconstruction of the Shigella T3SS transmembrane regions reveals 12-fold symmetry and novel features throughout. Nat Struct Mol Biol 16 477 485

6. BurghoutPBeckersFde WitEvan BoxtelRCornelisGR 2004 Role of the pilot protein YscW in the biogenesis of the YscC secretin in Yersinia enterocolitica. J Bacteriol 186 5366 5375

7. CragoAMKoronakisV 1998 Salmonella InvG forms a ring-like multimer that requires the InvH lipoprotein for outer membrane localization. Mol Micro 30 47 56

8. HardieKRSeydelAGuilvoutIPugsleyAP 1996 The secretin-specific, chaperone-like protein of the general secretory pathway: Separation of proteolytic protection and piloting functions. Mol Micro 22 967 976

9. KooJTammamSKuSYSampaleanuLMBurrowsLL 2008 PilF Is an Outer Membrane Lipoprotein Required for Multimerization and Localization of the Pseudomonas aeruginosa Type IV Pilus Secretin. J Bacteriol 190 6961 6969

10. LarioPIPfuetznerRAFreyEACreaghLHaynesC 2005 Structure and biochemical analysis of a secretin pilot protein. EMBO J 24 1111 1121

11. ShevchikVECondemineC 1998 Functional characterization of the Erwinia chrysanthemi OutS protein, an element of a type II secretion system. Microbiology 144 1443219 3228

12. TrindadeMBJobVContreras-MartelCPelicicVDessenA 2008 Structure of a widely conserved type IV pilus biogenesis factor that affects the stability of secretin multimers. J Mol Biol 378 1031 1039

13. DerrickJ 2008 A Pilot Sheds Light on Secretin Assembly. Structure 16 1441 1442

14. KorotkovKVGonenTHolWGJ 2011 Secretins: dynamic channels for protein transport across membranes. Trends Biochem Sci 36 433 443

15. OkonMMoraesTFLarioPICreaghALHaynesCA 2008 Structural Characterization of the Type-III Pilot-Secretin Complex from Shigella flexneri. Structure 16 1544 1554

16. GolovanovAPBalasinghamSTzitzilonisCGoultBTLianLY 2006 The solution structure of a domain from the Neisseria meningitidis lipoprotein PiIP reveals a new beta-sandwich fold. J Mol Biol 364 186 195

17. SandkvistM 2001 Biology of type II secretion. Mol Microbiol 40 271 283

18. JohnsonTLAbendrothJHolWGJSandkvistM 2006 Type II secretion: from structure to function. FEMS Micro Letts 255 175 186

19. ShevchikVERobertBaudouyJCondemineG 1997 Specific interaction between OutD, an Erwinia chrysanthemi outer membrane protein of the general secretory pathway, and secreted proteins. EMBO J 16 3007 3016

20. KorotkovKVPardonESteyaertJHolWGJ 2009 Crystal Structure of the N-Terminal Domain of the Secretin GspD from ETEC Determined with the Assistance of a Nanobody. Structure 17 255 265

21. GuilvoutIChamiMEngelAPugsleyAPBayanN 2006 Bacterial outer membrane secretin PulD assembles and inserts into the inner membrane in the absence of its pilotin. EMBO J 25 5241 5249

22. ColeCBarberJDBartonGJ 2008 The Jpred 3 secondary structure prediction server. Nucl Acid Res 36 W197 W201

23. ShoemakerBAPortmanJJWolynesPG 2000 Speeding molecular recognition by using the folding funnel: The fly-casting mechanism. Proc Natl Acad Sci U S A 97 8868 8873

24. NouwenNRansonNSaibilHWolpensingerBEngelA 1999 Secretin PulD: Association with pilot PulS, structure, and ion-conducting channel formation. Proc Natl Acad Sci U S A 96 8173 8177

25. SampaleanuLMBonannoJBAyersMKooJTammamS 2009 Periplasmic Domains of Pseudomonas aeruginosa PilN and PilO Form a Stable Heterodimeric Complex. J Mol Biol 394 143 159

26. AbendrothJRiceAEMcLuskeyKBagdasarianMHolWGJ 2004 The crystal structure of the periplasmic domain of the type II secretion system protein EpsM from Vibrio cholerae: The simplest version of the ferredoxin fold. J Mol Biol 338 585 596

27. AbendrothJKregerACHolWGJ 2009 The dimer formed by the periplasmic domain of EpsL from the Type 2 Secretion System of Vibrio parahaemolyticus. J Str Biol 168 313 322

28. NakanoNKuboriTKinoshitaMImadaKNagaiH 2010 Crystal Structure of Legionella DotD: Insights into the Relationship between Type IVB and Type II/III Secretion Systems. PLOS Pathog 6 e1001129

29. LeimanPGBaslerMRamagopalUABonannoJBSauderJM 2009 Type VI secretion apparatus and phage tail-associated protein complexes share a common evolutionary origin. Proc Natl Acad Sci U S A 106 4154 4159

30. Garcia-HerreroAVogelHJ 2005 Nuclear magnetic resonance solution structure of the periplasmic signalling domain of the TonB-dependent outer membrane transporter FecA from Escherichia coli. Mol Micro 58 1226 1237

31. YipCKKimbroughTGFeliseHBVuckovicMThomasNA 2005 Structural characterization of the molecular platform for type III secretion system assembly. Nature 435 702 707

32. WorrallLJVuckovicMStrynadkaNCJ 2010 Crystal structure of the C-terminal domain of the Salmonella type III secretion system export apparatus protein InvA. Protein Sci 19 1091 1096

33. FriesMIhrigJBrocklehurstKShevchikVEPickersgillRW 2007 Molecular basis of the activity of the phytopathogen pectin methylesterase. EMBO J 26 3879 3887

34. LoginFHShevchikVE 2006 The single transmembrane segment drives self-assembly of OutC and the formation of a functional type II secretion system in Erwinia chrysanthemi. J Biol Chem 281 33152 33162

35. BerglerHAbrahamDAschauerHTurnowskyF 1994 Inhibition of lipid biosynthesis induces the expression of the pspa gene. Microbiology 140 1937 1944

36. BouleyJCondemineGShevchikVE 2001 The PDZ domain of OutC and the N-terminal region of OutD determine the secretion specificity of the type II out pathway of Erwinia chrysanthemi. J Mol Biol 308 205 219

37. LeslieAGW 2006 The integration of macromolecular diffraction data. Acta Cryst D62 48 57

38. EvansP 2006 Scaling and assessment of data quality. Acta Cryst D62 72 82

39. AdamsPDAfoninePVBunkocziGChenVBDavisIW 2010 PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Cryst D66 213 221

40. EmsleyPLohkampBScottWGCowtanK 2010 Features and development of Coot. Acta Cryst D66 486 501

41. HolmLKaariainenSRosenstromPSchenkelA 2008 Searching protein structure databases with DaliLite v.3. Bioinformatics 24 2780 2781

42. LarkinMABlackshieldsGBrownNPChennaRMcGettiganPA 2007 Clustal W and clustal X version 2.0. Bioinformatics 23 2947 2948

43. BaxAGrishaevA 2005 Weak alignment NMR: a hawk-eyed view of biomolecular structure. Curr Opin Struct Biol 15 563 570

44. SreeramaNVenyaminovSYWoodyRW 2000 Estimation of protein secondary structure from circular dichroism spectra: Inclusion of denatured proteins with native proteins in the analysis. Anal Biochem 287 243 251

45. MartinSRSchilstraMJ 2008 Circular dichroism and its application to the study of biomolecules. Biophysical Tools for Biologists: Vol 1 in Vitro Techniques San Diego Elsevier Academic Press Inc 263 293

46. WeissMS 2001 Global indicators of X-ray data quality. J Appl Cryst 34 130 135