Modular organization of the AIDA autotransporter translocator: the N-terminal beta1-domain is surface-exposed and stabilizes the transmembrane beta2-domain

Antonie Van Leeuwenhoek. 2001 Oct;80(1):19-34. doi: 10.1023/a:1012084325728.

Abstract

The adhesin involved in diffuse adherence (AIDA-I) of the diarrhoeagenic Escherichia coli strain 2787 (O126:H27) is synthesized as a precursor molecule. This pre-pro-protein is N- and C-terminally processed to generate three distinct domains, which are characteristic for autotransporter secretion systems in Gram-negative bacteria: the N-terminal pre-peptide, the alpha-domain and the C-terminal beta-domain. The outer membrane-integrated beta-domain (AIDAC) is responsible for the surface-presentation of the alpha-domain (AIDA-I) and is thus termed 'translocator'. Characterization of extracted N-terminally truncated forms and of in vitro refolded proteins revealed a core structure at the C-terminus of the translocator which was found to be very stable even in the presence of SDS. Denaturation occurs only after additional incubation at temperatures above 80 degrees C. Reporter-epitope insertions were used to analyze the location of regions of membrane-integrated AIDAC relative to the membrane. The modified topological model developed for the AIDA translocator suggests the N-terminal domain (beta1) encompasses approximately 10 kDa to represent a completely surface-exposed segment while the C-terminal compact core domain (beta2) remains integrated in the membrane as a beta-barrel-like structure. Though the beta2-core structure alone harbours all the information for the outer membrane integration of AIDAC it is additionally stabilized by the beta1-domain. Access to large amounts of complete as well as truncated AIDAC proteins facilitated the study of protein folding by CD and fluorescence spectroscopy. A potential pore forming activity of the translocator using the completely refolded AIDAC or the beta2-core in black-lipid membranes could not be demonstrated.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adhesins, Escherichia coli / chemistry*
  • Adhesins, Escherichia coli / genetics
  • Adhesins, Escherichia coli / metabolism*
  • Base Sequence
  • Cell Membrane / metabolism*
  • Circular Dichroism
  • Endopeptidases / metabolism
  • Escherichia coli / chemistry
  • Escherichia coli / genetics
  • Escherichia coli / metabolism*
  • Hot Temperature
  • Models, Molecular
  • Molecular Sequence Data
  • Protein Folding
  • Protein Precursors / chemistry
  • Protein Precursors / genetics
  • Protein Precursors / metabolism
  • Protein Structure, Tertiary
  • Recombinant Fusion Proteins / chemistry
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism
  • Sequence Deletion
  • Spectrometry, Fluorescence

Substances

  • AIDA-I protein, E coli
  • Adhesins, Escherichia coli
  • Protein Precursors
  • Recombinant Fusion Proteins
  • Endopeptidases