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

Crespo, Maria D.; Puorger, Chasper; Schärer, Martin A.; Eidam, O.; Grütter, Markus G.; Capitani, Guido; Glockshuber, Rudi

doi:10.1038/nchembio.1019

Cited by 47 publications

(74 citation statements)

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“…This is likely an important feature given that several key substrates are oxidized by DsbA independently of folding, which occurs only after oxidation and with the assistance of a specific folding chaperone. For these substrates, which include the structural and adhesive subunits of the Fim and Pap pilus systems, random diffusion rather than folding drives the cognate cysteines together (1,43). Because this process is not guided by a funneled energy landscape, unlike folding, it may take longer or have greater variability in the time required for oxidation.…”

Section: Figurementioning

confidence: 99%

Monitoring Oxidative Folding of a Single Protein Catalyzed by the Disulfide Oxidoreductase DsbA

Kahn

Fernández

Pérez-Jiménez

2015

Journal of Biological Chemistry

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Background:The enzyme DsbA is essential for production of disulfide-bonded proteins in E. coli. Results: The folding, misfolding, and release kinetics of a substrate interacting with DsbA are determined from single molecule observations. Conclusion: DsbA is much more effective than its eukaryotic counterpart. Significance: Previous mechanistic models are generalized while providing insight into the kinetic parameters that influence oxidative folding outcomes.

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Section: Figurementioning

confidence: 99%

Monitoring Oxidative Folding of a Single Protein Catalyzed by the Disulfide Oxidoreductase DsbA

Kahn

Fernández

Pérez-Jiménez

2015

Journal of Biological Chemistry

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“…In the periplasm the disulphide bonds of the subunits are formed by the disulphide oxidoreductase DsbA. A two-domain 'L' shaped chaperone, FimC/PapD, then collects the subunit, binding mainly via its N-terminal domain, rejecting those disulphide misfolds for peptidic recycling [13,21]. The complete subunit folding processes have been examined in the type 1 rod subunit, FimA [21].…”

Section: The Individual Subunits In the Periplasmmentioning

confidence: 99%

Reprint of “Biogenesis and adhesion of type 1 and P pili”

Lillington¹,

Geibel²,

Waksman³

2015

Biochimica et Biophysica Acta (BBA) - General Subjects

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“…The peaks corresponding to oxidized and reduced ClyA were integrated, and the data were evaluated according to pseudo first-order kinetics. The reaction between monomeric ClyA red (4 M) and oxidized DsbA ox (86 M, corresponding to the periplasmic DsbA concentration in E. coli (24)) at pH 7.3 and 37°C, was analyzed in the same way.…”

Section: Methodsmentioning

confidence: 99%

Soluble Oligomers of the Pore-forming Toxin Cytolysin A from Escherichia coli Are Off-pathway Products of Pore Assembly

Roderer

Benke

Schuler

et al. 2016

Journal of Biological Chemistry

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The ␣-pore-forming toxin Cytolysin A (ClyA) is responsible for the hemolytic activity of various Escherichia coli and Salmonella enterica strains. Soluble ClyA monomers spontaneously assemble into annular dodecameric pore complexes upon contact with membranes or detergent. At ClyA monomer concentrations above ϳ100 nM, the rate-limiting step in detergent-or membrane-induced pore assembly is the unimolecular reaction from the monomer to the assembly-competent protomer, which then oligomerizes rapidly to active pore complexes. In the absence of detergent, ClyA slowly forms soluble oligomers. Here we show that soluble ClyA oligomers cannot form dodecameric pore complexes after the addition of detergent and are hemolytically inactive. In addition, we demonstrate that the natural cysteine pair Cys-87/Cys-285 of ClyA forms a disulfide bond under oxidizing conditions and that both the oxidized and reduced ClyA monomers assemble to active pores via the same pathway in the presence of detergent, in which an unstructured, monomeric intermediate is transiently populated. The results show that the oxidized ClyA monomer assembles to pore complexes about one order of magnitude faster than the reduced monomer because the unstructured intermediate of oxidized ClyA is less stable and dissolves more rapidly than the reduced intermediate. Moreover, we show that oxidized ClyA forms soluble, inactive oligomers in the absence of detergent much faster than the reduced monomer, providing an explanation for several contradictory reports in which oxidized ClyA had been described as inactive. Pore-forming toxins (PFTs)2 exist in different orders of bacteria and eukaryotes and cause various human diseases (1). Some of the most potent bacterial toxins are PFTs, such as anthrax toxin (2) and cytolysin from Vibrio cholerae (3). A common feature of all PFTs is the conversion from a soluble, monomeric form into a membrane-embedded oligomeric pore complex (1). The membrane-spanning region of the pore can be formed either by ␣-helices or ␤-strands; therefore, PFTs are classified as ␣-PFTs and ␤-PFTs (4).The 34-kDa PFT Cytolysin A (ClyA, also termed Hemolysin E (HlyE)) is an ␣-PFT existing in various Escherichia coli and Salmonella enterica strains (5-10). The structure of the annular, dodecameric pore complex of E. coli ClyA has been solved to atomic resolution (Protein Data Bank ID: 2WCD) (11). The pore subunit (protomer) shows major structural differences when compared with the soluble ClyA monomer (Protein Data Bank ID: 1QOY) (12): The soluble, monomeric form of ClyA consists of a large tail domain with four long ␣-helices and one short ␣-helix (␣-helices A, B, C, F, and G; residues 2-159 and 206 -303) and a head domain (residues 160 -205) with a central, hydrophobic ␤-hairpin (the "␤-tongue," residues 185-195) flanked by two short ␣-helices (␣-helices D and E) (Fig. 1). The tail domain contains a conserved cysteine pair (Cys-87 and Cys-285 in ␣-helix B and G, respectively) that form a disulfide bond (12-14). During pore formation, ClyA undergo...

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

Cited by 47 publications

References 50 publications

Monitoring Oxidative Folding of a Single Protein Catalyzed by the Disulfide Oxidoreductase DsbA

Monitoring Oxidative Folding of a Single Protein Catalyzed by the Disulfide Oxidoreductase DsbA

Reprint of “Biogenesis and adhesion of type 1 and P pili”

Soluble Oligomers of the Pore-forming Toxin Cytolysin A from Escherichia coli Are Off-pathway Products of Pore Assembly

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