Influence of the conserved disulphide bond, exposed to the putative binding pocket, on the structure and function of the immunoglobulin-like molecular chaperone Caf1M of Yersinia pestis

Abstract

The Yersinia pestis protein Caf1M is a typical representative of a subfamily of periplasmic molecular chaperones with characteristic structural and functional features, one of which is the location of two conserved cysteine residues close to the putative binding pocket. We show that these residues form a disulphide bond, the reduction and alkylation of which significantly increases the dissociation constant of the Caf1M-Caf1 (where Caf 1 is a polypeptide subunit of the capsule) complex [from a K d of (4.77p0.50)i10 −* M for the intact protein to one of (3.68p0.68)i10 −) M for the modified protein]. The importance of the disulphide bond for the formation of functional Caf1M in i o was demonstrated using an Escherichia coli dsbA mutant carrying the Y. pestis f1 operon. In accordance with the CD and fluorescence measurements, the disulphide bond is not important for maintenance of the overall structure of the Caf1M molecule,

Characterization of Two Homologous Disulfide Bond Systems Involved in Virulence Factor Biogenesis in Uropathogenic Escherichia coli CFT073

“…Other members of the PapD-like chaperone superfamily have been described from E. coli and other organisms (20,40). For example, the Caf1M chaperone of capsule in Yersinia pestis is the prototypic member of the FGL subfamily of molecular chaperones and contains two invariable cysteine residues that form a disulfide bond in the subunit binding pocket (53,54). Formation of this disulfide bond is catalyzed by DsbA and is required for the appropriate folding of Caf1M and for binding to the Caf1 capsule subunit of Y. pestis (53).…”

“…For example, the Caf1M chaperone of capsule in Yersinia pestis is the prototypic member of the FGL subfamily of molecular chaperones and contains two invariable cysteine residues that form a disulfide bond in the subunit binding pocket (53,54). Formation of this disulfide bond is catalyzed by DsbA and is required for the appropriate folding of Caf1M and for binding to the Caf1 capsule subunit of Y. pestis (53). Based on our results, it is possible that DsbL may also be capable of catalyzing disulfide bond formation in Caf1M and other members of the FGL subfamily of chaperones.…”

Molecular Aspects of Biogenesis of Escherichia coli Dr Fimbriae: Characterization of DraB-DraE Complexes

“…The E. coli BL21(DE3) and B178(DE3) htrA63 strains transformed with appropriate plasmids were typically cultivated to an A 600 of 0.4 in Luria-Bertani medium supplemented with 20 g of kanamycin per ml, and protein expression was induced by adding isopropyl-␤-D-thiogalactopyranoside (IPTG) to a final concentration of 0.5 mM for 2 h. Then the cultures were harvested by centrifugation, and periplasmic fractions containing the target proteins were extracted by the osmotic shock procedure (41). To purify complexes of the DraB chaperone with DraE oligomers, the periplasmic fraction obtained from 1 liter of culture was dialyzed overnight against buffer A containing 10 mM NaCl and 20 mM Tris-HCl (pH 7.0).…”

Gram-positive DsbE Proteins Function Differently from Gram-negative DsbE Homologs

“…DsbA is reoxidized by the transmembrane protein DsbB, which is in turn oxidized by components of the electron transport pathway (12,13). Dsb proteins, in particular DsbA, have been shown to be involved in virulence in toxinsecreting Gram-negative bacteria such as Vibrio cholerae (14,15), Yersinia pestis (16), Shigella sp. (17), and E. coli (18).…”