A conserved region within the Bordetella pertussis autotransporter BrkA is necessary for folding of its passenger domain

Mol Microbiol. 2003 Mar;47(5):1367-83. doi: 10.1046/j.1365-2958.2003.03377.x.

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.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Bacterial Adhesion
  • Bacterial Outer Membrane Proteins / chemistry*
  • Bacterial Outer Membrane Proteins / genetics
  • Bacterial Outer Membrane Proteins / physiology
  • Bordetella pertussis / chemistry*
  • Bordetella pertussis / genetics
  • Circular Dichroism
  • Escherichia coli / metabolism
  • Escherichia coli Proteins
  • Genetic Complementation Test
  • HeLa Cells
  • Humans
  • Membrane Proteins
  • Models, Molecular
  • Molecular Sequence Data
  • Peptide Hydrolases
  • Porins / genetics
  • Porins / metabolism
  • Protein Conformation
  • Protein Folding*
  • Protein Sorting Signals
  • Protein Structure, Tertiary
  • Sequence Alignment
  • Sequence Homology, Amino Acid
  • Structure-Activity Relationship

Substances

  • Bacterial Outer Membrane Proteins
  • BrkA protein, Bordetella pertussis
  • Escherichia coli Proteins
  • Membrane Proteins
  • Porins
  • Protein Sorting Signals
  • ompT protein, E coli
  • Peptide Hydrolases