The N-terminal beta-barrel domain of the Escherichia coli K88 periplasmic chaperone FaeE determines fimbrial subunit recognition and dimerization.

The K88 periplasmic chaperone FaeE is a homodimer, whereas the K99 chaperone FanE is a monomer. The structural requirements for dimerization of the K88 fimbrial periplasmic chaperone and for fimbrial subunit-binding specificity were investigated by analysis of mutant chaperones. FaeE contains a C-terminal extension of 19 amino acid residues when compared to FanE and most other fimbrial chaperones. A C-terminal truncate of the K88 chaperone FaeE was constructed that lacked 19 C-terminal amino acid residues. Expression and complementation experiments revealed that this C-terminal shortened chaperone was still functional in binding the K88 major subunit FaeG and K88 biosynthesis. Two hybrid chaperones were constructed. Each hybrid protein contained one beta-barrel domain of FaeE and the other beta-barrel domain of FanE (Fae/FanE or Fan/FaeE, respectively). Expression and complementation experiments revealed that the Fae/FanE but not the Fan/FaeE hybrid chaperone was functional in the formation of K88 fimbriae. The Fan/FaeE hybrid chaperone was active in the bio-synthesis of K99 fimbriae. The truncated FaeE mutant chaperone and the hybrid Fae/FanE chaperone were able to form stable periplasmic protein complexes with the K88 major fimbrial subunit FaeG. Cross-linking experiments suggested that the C-terminal shortened chaperone and the Fae/FanE hybrid chaperone were homodimers, as is the wild-type K88 chaperone. Altogether, the data suggested that the N-terminal beta-barrel domain of a fimbrial chaperone determines subunit specificity. In the case of the K88 periplasmic chaperone, this N-terminal domain also determines dimerization of the protein.

The Escherichia coli K99 periplasmic chaperone FanE is a monomeric protein.

The monomeric or dimeric nature of the K99 periplasmic chaperone FanE was examined. The gene encoding FanE was subcloned in a pINIIIA1 derivative expression vector. A complementation experiment showed that the subcloned FanE was biologically functional. The protein was purified from the periplasm of cells harbouring the constructed plasmid. Automated Edman degradation experiments confirmed the predicted N-terminal amino acid sequence of FanE. A polyclonal mouse antiserum was raised against the FanE chaperone. The monomeric or oligomeric nature of the protein in the periplasm was studied by gel filtration, immunoblotting and chemical cross-linking experiments. The results indicated that FanE is a monomeric protein, in contrast to the K88 periplasmic chaperone.