title: Stepwise folding of an autotransporter passenger domain is not essential for its secretion.
abstract: Autotransporters are a superfamily of virulence proteins produced by Gram-negative bacteria. They consist of an N-terminal β-helical domain ("passenger domain") that is secreted into the extracellular space and a C-terminal β-barrel domain ("β-domain") that anchors the protein to the outer membrane. Because the periplasm lacks ATP, vectorial folding of the passenger domain in a C-to-N-terminal direction has been proposed to drive the secretion reaction. Consistent with this hypothesis, mutations that disrupt the folding of the C terminus of the passenger domain of the Escherichia coli O157:H7 autotransporter EspP have been shown to cause strong secretion defects. Here, we show that point mutations introduced at specific locations near the middle or N terminus of the EspP β-helix that perturb folding also impair secretion, but to a lesser degree. Surprisingly, we found that even multiple mutations that potentially abolish the stability of several consecutive rungs of the β-helix only moderately reduce secretion efficiency. Although these results provide evidence that the free energy derived from passenger domain folding contributes to secretion efficiency, they also suggest that a significant fraction of the energy required for secretion is derived from another source.
authors.affiliation: From the Genetics and Biochemistry Branch, NIDDK, National Institutes of Health, Bethesda, Maryland 20892.
authors.forename: Wanyoike
authors.initials: W
authors.lastname: Kang'ethe
authors.forename: Harris D
authors.initials: HD
authors.lastname: Bernstein
mesh_terms: D019943:Amino Acid Substitution; D002462:Cell Membrane / Q000378:metabolism; D019453:Escherichia coli O157 / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D017510:Protein Folding; D055550:Protein Stability; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D012697:Serine Endopeptidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D029968:Escherichia coli Proteins; C107189:EspP protein, E coli; D012697:Serine Endopeptidases
keywords: Autotransporter; Escherichia coli; Membrane Proteins; Protein Folding; Protein Translocation; Virulence Factors