Mechanism of fibre assembly through the chaperone–usher pathway

Abstract

The chaperone-usher pathway directs the formation of adhesive surface fibres in numerous pathogenic Gram-negative bacteria. The fibres or pili consist exclusively of protein subunits that, before assembly, form transient complexes with a chaperone in the periplasm. In these chaperone:subunit complexes, the chaperone donates one b-strand to complete the imperfect immunoglobulin-like fold of the subunit. During pilus assembly, the chaperone is replaced by a polypeptide extension of another subunit in a process termed 'donor strand exchange' (DSE). Here we show that DSE occurs in a concerted reaction in which a chaperone-bound acceptor subunit is attacked by another chaperone-bound donor subunit. We provide evidence that efficient DSE requires interactions between the reacting subunits in addition to those involving the attacking donor strand. Our results indicate that the pilus assembly platforms in the outer membrane, referred to as ushers, catalyse fibre formation by increasing the effective concentrations of donor and acceptor subunits.

Quality control of disulfide bond formation in pilus subunits by the chaperone FimC

“…Recently, we reported the NMR structure of a designed, selfcomplemented FimA variant (FimAa) 28 , in which FimA is artificially extended at its C terminus by a hexaglycine linker followed by the FimA donor strand segment (residues [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20]. FimAa has the same slow, spontaneous folding rate as wild-type FimA (1.6-h folding half-life) and adopts a conformation in which the C-terminal copy of the donor strand is incorporated into the tertiary structure in an antiparallel orientation relative to the FimA F strand, which corres ponds to the expected donor strand insertion pattern in the quaternary structure of the pilus rod 28 .…”

“…To unravel the structural basis of the at least 10 4 -fold acceleration of FimA folding upon binding to FimC, we solved the X-ray structure of the FimC-FimA complex at 2.5-Å resolution and compared it with the structure of FimA a . To prevent formation of FimA homopolymers 28 , we used an N-terminally truncated FimA variant (FimA t ) lacking residues 1-17 of FimA 14,28 and bearing an uncleaved methionine at the N terminus. refers to the first (chains A and B) of the two complexes found in the asymmetric unit.…”

“…1b). The chaperonesubunit complexes then diffuse to the outer membrane usher FimD, where the N-terminal extension of the incoming subunits replaces the donor strand of the chaperone on the previously incorporated subunit at the growing pilus end in a reaction termed donor strand exchange 7,11,[14][15][16] . In contrast to the 'parallel' orientation of the donor strand in FimC-subunit complexes, the donor strand complementing the fold of neighboring subunits in the pilus has an antiparallel orientation relative to the F strand of the acceptor subunit.…”

Both ATPase Domains of ClpA Are Critical for Processing of Stable Protein Structures

“…Each FimA subunit has an unstructured N-terminal extension (ϭ donor strand) that binds to and completes the fold of the adjacent FimA subunit. FimAta is an artificial FimA variant that is N-terminally truncated and C-terminally elongated by a short linker followed by the donor strand, leading to extensive stabilization of the C terminus through "self-complementation" (22) (Fig. 2d).…”

“…-repressor92C-ssrA was purified by ion exchange chromatography (DEAE-Sephacel) before being applied to a heparin-Sepharose column followed by size exclusion chromatography (Superdex 75). His 6 -TEV-FimAta-li-ssrA was purified as described previously (22) using chelating Ni 2ϩ chromatography instead of anion exchange chromatography as the last step of purification. Correct folding of purified His 6 -TEV-FimAta-li-ssrA was confirmed by circular dichroism spectroscopy.…”