Structure, Folding and Stability of FimA, the Main Structural Subunit of Type 1 Pili from Uropathogenic Escherichia coli Strains

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

“…1), confirmed that no major structure formation in FimA U ox is required for recognition by FimC. The reported half-life of 1.6 h (k f = 0.425 h −1 ) of spontaneous FimA U ox folding 28 was confirmed, and spontaneous folding of FimA U red proceeded even six times slower with a half-life of 9.9 h (k f = 0.070 h −1 ) (Supplementary Fig. 1 Figure 2c shows that FimA U ox formed a complex with FimC within the dead time of the experiment (about 1 min) after rapid dilution of FimA U ox into native buffer containing a two-fold excess of FimC.…”

“…As FimA lacks tryptophan residues, we first tested whether binding of FimA can be detected via a tryptophan fluorescence change in FimC. Figure 2a shows that addition of a two-fold excess of FimA U ox to FimC under native conditions resulted in a ~25% 28 , we concluded that the decrease in FimC fluorescence upon addition of FimA U ox results from binding of the unfolded, disulfide-intact subunit and/or subunit folding on the surface of FimC and that FimC is unable to recognize unfolded subunits lacking the disulfide bond. The lack of secondary structure in both FimA U ox and FimA U red after the onset of refolding, verified with far-UV CD spectroscopy ( Supplementary Fig.…”

“…4b and Supplementary Table 2). We interpret the slow-folding species as an in vitro folding artifact caused by the cis-to-trans isomerization of non-native cis-prolyl peptide bonds that accumulate in FimA U ox upon longterm incubation in denaturant; as the only two prolyl peptide bonds (isoleucine-proline and threonine-proline) in FimA are in the trans conformation in the native state 28 , 20% of FimA U ox molecules with at least one non-native cis-prolyl peptide bond is indeed predicted for unfolded FimA on the basis of studies on unstructured model peptides 33 . In addition, the slower phase in Figure 4b shows a concentration-independent half-life of 18 s (Supplementary Table 2), which is in the typical range for a prolyl cis-to-trans isomerization at 25 °C 34 .…”

Molecular mechanism of bacterial type 1 and P pili assembly

“…type 1 and P pili, respectively-is embedded into the OM via the β-barrel assembly machinery (BAM complex) [9,10]. The periplasmic chaperones FimC and PapD promote subunit/pilin folding as they are released by the Sec translocon and target pilus subunits to their respective usher [11,12].…”

“…The pilin is stable only when the cognate chaperone completes the pilin's Ig-like fold by occupying the pilin's groove with part of its own G1 strand, a process called donor strand complementation (DSC) [31][32][33] (figure 2). DSC stabilizes pilus subunits as they emerge from the Sec translocon in the IM, promotes their folding, and also prevents their premature self-polymerization in the periplasm [12,34]. During DSC, the chaperone's G1 strand runs parallel to strand F of the pilus subunit, and, thus, the non-covalently assembled Ig-fold that results is non-canonical.…”