Off‐pathway assembly of fimbria subunits is prevented by chaperone CfaA of CFA/I fimbriae from enterotoxigenic E. coli

Summary The assembly of the class 5 colonization factor antigen I (CFA/I) fimbriae of enterotoxigenic E. coli was proposed to proceed via the alternate chaperone‐usher pathway. Here, we show that in the absence of the chaperone CfaA, CfaB, the major pilin subunit of CFA/I fimbriae, is able to spontaneously refold and polymerize into cyclic trimers. CfaA kinetically traps CfaB to form a metastable complex that can be stabilized by mutations. Crystal structure of the stabilized complex reveals distinctive interactions provided by CfaA to trap CfaB in an assembly competent state through donor‐strand complementation (DSC) and cleft‐mediated anchorage. Mutagenesis indicated that DSC controls the stability of the chaperone‐subunit complex and the cleft‐mediated anchorage of the subunit C‐terminus additionally assist in subunit refolding. Surprisingly, over‐stabilization of the chaperone‐subunit complex led to delayed fimbria assembly, whereas destabilizing the complex resulted in no fimbriation. Thus, CfaA acts predominantly as a kinetic trap by stabilizing subunit to avoid its off‐pathway self‐polymerization that results in energetically favorable trimers and could serve as a driving force for CFA/I pilus assembly, representing an energetic landscape unique to class 5 fimbria assembly. The colonization factor antigen I (CFA/I) pili are archetypal of a group of human‐specific enterotoxigenic E. coli (ETEC) and are assembled via the alternate chaperone‐usher pathway. Here, we present the structure of the chaperone CfaA in complex with subunit CfaB and demonstrate that the role of CfaA in CFA/I pilus assembly is to avoid off pathway self‐polymerization of the subunit CfaB (dotted arrows).