Structural and Functional Significance of the FGL Sequence of the Periplasmic Chaperone Caf1M of Yersinia pestis

Abstract

The periplasmic molecular chaperone Caf1M of Yersinia pestis is a typical representative of a subfamily of specific chaperones involved in assembly of surface adhesins with a very simple structure. One characteristic feature of this Caf1M-like subfamily is possession of an extended, variable sequence (termed FGL) between the F1 and subunit binding G1 β-strands. In contrast, FGS subfamily members, characterized by PapD, have a short F1-G1 loop and are involved in assembly of complex pili. To elucidate the structural and functional significance of the FGL sequence, a mutant Caf1M molecule (dCaf1M), in which the 27 amino acid residues between the F1 and G1 β-strands had been deleted, was constructed. Expression of the mutated caf1M in Escherichia coli resulted in accumulation of high levels of dCaf1M. The far-UV circular dichroism spectra of the mutant and wild-type proteins were indistinguishable and exhibited practically the same temperature and pH dependencies. Thus, the FGL sequence of Caf1M clearly does not contribute significantly to the stability of the protein conformation. Preferential cleavage of Caf1M by trypsin at Lys-119 confirmed surface exposure of this part of the FGL sequence in the isolated chaperone and periplasmic chaperone-subunit complex. There was no evidence of surface-localized Caf1 subunit in the presence of the Caf1A outer membrane protein and dCaf1M. In contrast to Caf1M, dCaf1M was not able to form a stable complex with Caf1 nor could it protect the subunit from proteolytic degradation in vivo. This demonstration that the FGL sequence is required for stable chaperone-subunit interaction, but not for folding of a stable chaperone, provides a sound basis for future detailed molecular analyses of the FGL subfamily of chaperones.

Secretion of Recombinant Proteins via the Chaperone/Usher Pathway in Escherichia coli

“…PapD contains two domains, each with a ␤-barrel and an immunoglobulin (Ig)-like fold (8). The caf gene cluster that produces and assembles the capsular F1 (Caf1) antigen of Yersinia pestis is the best-characterized representative of the second family (2,6,7,12,22,37). The genes encode a 26.5-kDa periplasmic chaperone (Caf1M) (7) and a 90.4-kDa outer membrane protein (Caf1A) (12), which together can mediate the surface assembly of Caf1 antigen (6) in recombinant E. coli cells (2,13).…”

“…The caf gene cluster that produces and assembles the capsular F1 (Caf1) antigen of Yersinia pestis is the best-characterized representative of the second family (2,6,7,12,22,37). The genes encode a 26.5-kDa periplasmic chaperone (Caf1M) (7) and a 90.4-kDa outer membrane protein (Caf1A) (12), which together can mediate the surface assembly of Caf1 antigen (6) in recombinant E. coli cells (2,13). Caf1Mlike periplasmic chaperones are characterized by an extended variable sequence between the proposed F1 and G1 ␤-strands, a disulfide bond connecting these two strands, and an accessory N-terminal sequence (2,36,37).…”

“…The genes encode a 26.5-kDa periplasmic chaperone (Caf1M) (7) and a 90.4-kDa outer membrane protein (Caf1A) (12), which together can mediate the surface assembly of Caf1 antigen (6) in recombinant E. coli cells (2,13). Caf1Mlike periplasmic chaperones are characterized by an extended variable sequence between the proposed F1 and G1 ␤-strands, a disulfide bond connecting these two strands, and an accessory N-terminal sequence (2,36,37). Together, these three features may form an extension to the binding domain, which is important for chaperone function (2,22,37).…”

“…Caf1Mlike periplasmic chaperones are characterized by an extended variable sequence between the proposed F1 and G1 ␤-strands, a disulfide bond connecting these two strands, and an accessory N-terminal sequence (2,36,37). Together, these three features may form an extension to the binding domain, which is important for chaperone function (2,22,37). In contrast to pap-like gene clusters, all members of caf-like gene clusters are involved in the assembly of structures with a simple composition and a less rigid structure (fibrillae or capsule-like morphology) (9,36).…”

“…This donor strand complementation interaction between periplasmic chaperone and structural subunit appears to occur at the level of the inner membrane and appears to be required for correct folding prior to release of the subunit from the inner membrane (10). Mutagenesis studies have provided strong evidence that the Caf1M chaperone uses a similar ␤-donor strand complementation mechanism to promote correct folding of Caf1 subunit at the inner membrane, although in this case the chaperone-subunit interaction is mediated by a particularly long, alternating hydrophobic extension to the chaperone G1 ␤-strand (2,22).…”