A variant of CfaE, donor strand complemented CfaE (dscCfaE), containing a C-terminal hairpin linker followed by the first 19 amino acid residues derived from the N-terminus of the major fimbrial subunit CfaB was purified to homogeneity. The dscCfaE protein was readily crystallized and the structure was determined. The dscCfaE molecule consists of two domains of roughly equal size. The N-terminal domain of CfaE is referred to as the adhesin domain (CfaEad) and is represented in the structure from residues A23 to D200. It consists of one anti-parallel beta-sheet (Sheet 1) and one mixed beta-sheet (Sheet 2). The C-terminal domain immediately follows the short three-residue linker (K201-G202-N203) and mediates attachment of the adhesive subunit to the main body of the fimbria. It is therefore termed the pilin domain (CfaEpd). The pilin domain folds into a beta-sandwich with a topology reminiscent of the adhesin domain. Both CfaEad and CfaEpd beta-structures display a topology that resembles the v-type Ig fold with nine beta-strands. In order to understand how the major and minor subunits are assembled into a CFA/I fimbria, we further engineered the donor strand complemented CfaEB complex (dscCfaEB) construct, which was expressed in E. coli. The recombinant dscCfaEB protein was purified and crystallized. The crystal structure of the CfaEB complex was determined, providing structural information on not only the major subunit CfaB, but also the geometry of the connection between the major and minor subunit. In addition to the CfaEB complex, we also determined crystal structures for the major-major subunit complexes CfaBB and CfaBBB, providing a basis for constructing a model of CFA/I pilus consistent with EM reconstructions of purified CFA/I pilus. Located at the upper surface of CfaEad distal to the CfaEpd, R181, which was previously known to be important for binding, is found in a positively charged depression and surrounded by a cluster of residues that are highly conserved in the Class 5 fimbrial adhesins, including residues from three different loops (i.e., B-C, D-E, and F-G loops). This pocket thus appears to be a suitable location to which a negatively charged sialylated receptor might bind. To confirm the role of this domain, R67, which is adjacent to R181, was mutated to alanine (dscCfaE/R67A) and purified. Bead-adsorbed dscCfaE/R67A failed to agglutinate human erythrocytes, similar to our previous findings for the dscCfaE/R181A mutant. These results implicate the pocket anchored by these two residues as the putative receptor-binding domain. To determine the role in hemagglutination of individual residues in the neighborhood of R181, we introduced site-specific mutations into CfaE in the plasmid pMAM2, which encodes all components of the CFA/I and directs surface expression of mutant fimbriae with single site mutations of CfaE. Twelve such mutations involving residues that are either invariant (fully conserved) or are subclass-specific for Class 5 ETEC fimbrial adhesins were introduced. All positively charged residues (R181, R182, R67) are absolutely required for receptor binding and cluster together to form a positively charged center. The positively charged center of the binding pocket is surrounded by a band of subclass-specific residues. Mutations of those residues display altered interactions with red cells and several show discriminatory behavior to either human type-A or bovine red cell species. We elucidated, for the first time, atomic structures of an ETEC major pilin subunit, CfaB from colonization factor antigen I (CFA/I) fimbriae. These data are used to construct models for two morphological forms of CFA/I fimbriae that are both observed in vivo, the helical filament into which it is typically assembled, and an extended, unwound conformation. Modeling and corroborative mutational data indicate that proline isomerization is involved in the conversion between the helical and extended forms of CFA/I fimbriae. Our findings affirm the strong structural similarities seen between Class 5 fimbriae (from bacteria primarily causing gastrointestinal disease) and Class 1 pili (from bacteria that cause urinary, respiratory and other infections) in the absence of significant primary sequence similarity. They also suggest that morphological and biochemical differences between fimbrial types, regardless of class, provide structural specialization that facilitates survival of each bacterial pathotype in its preferred host microenvironment. Lastly, we present structural evidence for bacterial use of antigenic variation to evade host immune responses, in that residues occupying the predicted surface-exposed face of CfaB and related Class 5 pilins show much higher genetic sequence variability than the remainder of the pilin protein. More recently, we have also determined the crystal structure of CfaA, the chaperone component that is essential for assembly of CFA/I fimbriae. Pili assembled by the chaperone-usher pathway (CUP) require periplasmic chaperones that assist subunit folding, maintain their stability, and escort them to the site of bioassembly. Until now, CUP chaperones have been classified into two families, FGS and FGL, based on the short and long length of the subunit-interacting loops between its F1 and G1 beta-strands, respectively. CfaA is the chaperone for assembly of colonization factor antigen I (CFA/I) pili of enterotoxigenic E. coli (ETEC), a cause of diarrhea in travelers and young children. Here, the crystal structure of CfaA along with mutational analyses reveals some unique structural and functional features, leading us to propose a separate family for CfaA and closely related chaperones. Phenotypic changes resulting from mutations in regions unique to this chaperone family provides insight into their function. Moreover, those regions that distinguish CfaA from the FGL and FGS chaperone families appear to influence interactions with their cognate subunits and usher proteins during pilus assembly. Despite the lack of detailed understanding with respect to the energetics of fimbria assembly, periplasmic chaperones are thought to serve both as kinetic and energy traps by forming binary complexes with pilin subunits during subunit refolding, which prevents subunits from aggregating and subsequently drives their polymerization into ordered helical assemblies. The assembly of CFA/I fimbriae, an archetype in the family of class 5 fimbriae from enterotoxigenic E. coli, is assisted by the periplasmic chaperone CfaA, which belongs to a newly classified group of periplasmic chaperones. In this work, we show that unlike pilin subunits of other bacterial fimbriae, CfaB, the major pilin subunits of CFA/I, are stable in the absence of the chaperone and are able to spontaneously refold and polymerize into cyclic trimers both in vivo and in vitro. We further show that chaperone CfaA is able to kinetically trap CfaB to form a metastable complex. Stabilization of the CfaA and CfaB heterodimer by mutations led to the crystal structure of the complex, revealing distinctive interactions between CfaA and CfaB through donor-strand complementation and cleft-mediated anchorage. Mutagenesis indicated that donor-strand complementation controls the stability of the chaperone-subunit complex and the cleft-mediated anchorage of the subunit C-terminus appears to assist in subunit refolding. Surprisingly, over-stabilization of the chaperon-subunit complex led to delayed fimbria assembly, whereas destabilizing the complex resulted in no fimbriation. Thus, CfaA functions predominantly as a kinetic trap fine-tuned to avoid off-pathway subunit self-polymerization, which 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.
