title: A conserved region within the Bordetella pertussis autotransporter BrkA is necessary for folding of its passenger domain.
abstract: Autotransporter secretion represents a unique mechanism that Gram-negative bacteria employ to deliver proteins to their cell surface. BrkA is a Bordetella pertussis autotransporter protein that mediates serum resistance and contributes to adherence of the bacterium to host cells. BrkA is a 103 kDa protein that is cleaved to form a 73 kDa alpha-domain and a 30 kDa beta domain. The alpha domain, also referred to as the passenger domain, is responsible for the effector functions of the protein, whereas the beta domain serves as a transporter. In an effort to characterize BrkA secretion, we have shown that BrkA has a 42 amino acid signal peptide for transit across the cytoplasmic membrane, and a translocation unit made up of a short linker region fused to the beta-domain to ferry the passenger domain to the bacterial surface through a channel formed by the beta-domain. In this report, we provide genetic, biochemical and structural evidence demonstrating that a region within the BrkA passenger (Glu601-Ala692) is necessary for folding the passenger. This region is not required for surface display in the outer membrane protease OmpT-deficient Escherichia coli strain UT5600. However, a BrkA mutant protein bearing a deletion in this region is susceptible to digestion when expressed in E. coli strains expressing OmpT suggesting that the region is required to maintain a stable structure. The instability of the deletion mutant can be rescued by surface expressing Glu601-Ala692in trans suggesting that this region is acting as an intramolecular chaperone to effect folding of the passenger concurrent with or following translocation across the outer membrane.
authors.affiliation: Department of Microbiology and Immunology, University of British Columbia, 300-6174 University Blvd., Vancouver, BC, V6T 1Z3, Canada.
authors.forename: David C
authors.initials: DC
authors.lastname: Oliver
authors.forename: George
authors.initials: G
authors.lastname: Huang
authors.forename: Elena
authors.initials: E
authors.lastname: Nodel
authors.forename: Steve
authors.initials: S
authors.lastname: Pleasance
authors.forename: Rachel C
authors.initials: RC
authors.lastname: Fernandez
mesh_terms: D000595:Amino Acid Sequence; D001422:Bacterial Adhesion; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D001886:Bordetella pertussis / Q000737:chemistry* / Q000235:genetics; D002942:Circular Dichroism; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins; D005816:Genetic Complementation Test; D006367:HeLa Cells; D006801:Humans; D008565:Membrane Proteins; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010447:Peptide Hydrolases; D018272:Porins / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation; D017510:Protein Folding; D021382:Protein Sorting Signals; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship
chemical_list: D001425:Bacterial Outer Membrane Proteins; C090007:BrkA protein, Bordetella pertussis; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D018272:Porins; D021382:Protein Sorting Signals; C499588:ompT protein, E coli; D010447:Peptide Hydrolases