Untitled

Abstract

In the type III secretory system of bacterial pathogens, a large number of sequence-divergent but characteristically small (approximately 14-19 kDa), acidic (pI approximately 4-5) chaperone proteins have been identified. We present the 1.74 A resolution crystal structure of the Yersinia pseudotuberculosis chaperone SycE, whose action in promoting translocation of YopE into host macrophages is essential to Yersinia pathogenesis. SycE, a compact, globular dimer with a novel fold, has two large hydrophobic surface patches that may form binding sites for YopE or other type III components. These patches are formed by structurally key residues that are conserved among many chaperones, suggesting shared structural and functional relationships. A negative electrostatic potential covers almost the entire surface of SycE and is likely conserved in character, but not in detail, among chaperones. The structure provides the first structural insights into possible modes of action of SycE and type III chaperones in general.

Crystal Structure of the Yersinia enterocolitica Type III Secretion Chaperone SycT

“…Chaperones of class II assist the TTSS translocators, and the flagellar TTSS chaperones constitute class III according to this classification. Crystal structures of representatives of class IA (Salmonella SicP (13) and SigE (14); Yersinia SycE (15), SycH (16), and SycN/YscB (17); Escherichia coli CesT (14); Pseudomonas syringae AvrPphF ORF1 (18)), class IB (Shigella flexneri Spa15 (19)), and class III (Aquifex aeolicus FliS (20)) support this classification. The class IA chaperones SicP, SigE, SycE, CesT, and AvrPphF ORF1, although not similar on a sequence level, all form homodimers and share a common fold.…”

“…Finally, Trp-84 interacts with Phe-65 and Ala-71. The dimer interface of SycE, also mainly stabilized via hydrophobic contacts (15), is arranged differently, as depicted in Fig. 3D.…”

“…The only close homologue of SycT (56% identity) is found in the entomopathogenic Photorhabdus luminescens (28). SycT is 20% identical to Yersinia SycE, which has been structurally solved (15,29,30).…”

The Type III Secretion Chaperone SycE Promotes a Localized Disorder-to-Order Transition in the Natively Unfolded Effector YopE

“…This latter strain was generated through standard allelic exchange procedures, with the coding region for yopE being replaced by the coding region for kanR. 3 Wild-type and ⌬yopE Y. pseudotuberculosis were grown in BHI medium at 37°C until mid-log density, and type III secretion was induced by addition of sodium oxalate and MgCl 2 to final concentrations of 20 mM. After 3 h, bacteria were harvested by centrifugation (10,000 ϫ g, 20 min, 4°C), resuspended in 25 ml of binding buffer (supplemented with 1 mM phenylmethylsulfonyl fluoride and 0.5 mM E-64) per liter of bacterial culture, and lysed by sonication.…”

“…A large family of such chaperones exists, with individual chaperones functioning in the translocation of only a single or just a few corresponding effectors. Although chaperones have limited sequence identity (Յ 20%), their folds and modes of effector binding are well conserved (3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13). Chaperone dimers provide rigid and globular surfaces, around which effectors wrap an ϳ25-100-residue chaperone-binding (Cb) region in strikingly extended conformation (4,6,9,12,13).…”