Adhesive type 1 pili from uropathogenic Escherichia coli strains have a crucial role during infection by mediating the attachment to and potentially the invasion of host tissue. These filamentous, highly oligomeric protein complexes are assembled by the ‘chaperone–usher’ pathway1, in which the individual pilus subunits fold in the bacterial periplasm and form stoichiometric complexes with a periplasmic chaperone molecule that is essential for pilus assembly2,3,4. The chaperone subsequently delivers the subunits to an assembly platform (usher) in the outer membrane, which mediates subunit assembly and translocation to the cell surface5,6,7,8. Here we show that the periplasmic type 1 pilus chaperone FimC binds non-native pilus subunits and accelerates folding of the subunit FimG by 100-fold. Moreover, we find that the FimC–FimG complex is formed quantitatively and very rapidly when folding of FimG is initiated in the presence of both FimC and the assembly-competent subunit FimF, even though the FimC–FimG complex is thermodynamically less stable than the FimF–FimG complex. FimC thus represents a previously unknown type of protein-folding catalyst, and simultaneously acts as a kinetic trap preventing spontaneous subunit assembly in the periplasm.
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Impairment of the biomechanical compliance of P pili: a novel means of inhibiting uropathogenic bacterial infections?
European Biophysics Journal Open Access 12 January 2012
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We thank A. Bachmann and T. Kiefhaber for their assistance and discussions; A. Fritz for technical assistance; and R. Brunisholz for Edman sequencing and MALDI–TOF mass spectrometry. This work was supported by the Schweizerische Nationalfonds and the Swiss NCCR Program in Structural Biology.
The authors declare that they have no competing financial interests.
The data in this table are derived from the fluorescence traces of FimGt folding kinetics (Supplementary Fig. S3) in the presence of the chaperone FimCYY, the subunit FimFF and the donor strand peptides DSFimC, DSFimF, and DSFimG. (PDF 98 kb)
List of recombinant type1 pilus subunit constructs and synthetic donor strand peptides used in this study. (PDF 84 kb)
Spectroscopic properties and thermodynamic stability of FimGt. The data illustrate that the tryptophan fluorescence of FimGt changes upon folding and that FimGt can fold also in the absence of any donor strand. (PDF 91 kb)
Stopped flow fluorescence traces of FimGt refolding. Folding of FimGt is slow and neither the partner subunit FimFF nor synthetic donor strand peptides influence FimGt folding significantly. In contrast, the reaction catalysed by the chaperone FimC exhibits very rapid and complex folding kinetics. (PDF 294 kb)
The figure shows interrupted refolding experiments that follow the formation of native complexes between FimGt and various donor strand peptides. In addition, the determination of the apparent affinity of FimGt for the corresponding donor strand peptides is shown. (PDF 95 kb)
Comparison of FimC and the tryptophan-free variant FimCYY with respect to stability, spectroscopic properties and function. The results indicate that FimCYY retains the ability of FimC to form soluble complexes with subunits in vivo despite its lowered intrinsic stability. (PDF 254 kb)
Interrupted refolding was used to monitor the formation of native FimGt in the presence of both FimCYY and cyclophilin. Periplasmic cyclophilin does not abolish the slow phase in chaperone-assisted formation of native FimGt. (PDF 79 kb)
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Vetsch, M., Puorger, C., Spirig, T. et al. Pilus chaperones represent a new type of protein-folding catalyst. Nature 431, 329–333 (2004). https://doi.org/10.1038/nature02891
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