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Antonie van Leeuwenhoek

, Volume 80, Issue 1, pp 19–34 | Cite as

Modular organization of the AIDA autotransporter translocator: The N-terminal β1-domain is surface-exposed and stabilizes the transmembrane β2-domain

  • Marc P. J. Konieczny
  • Inga Benz
  • Britta Hollinderbäumer
  • Christina Beinke
  • Michael NiederweisEmail author
  • M. Alexander Schmidt
Article

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 α-domain and the C-terminal β-domain. The outer membrane-integrated β-domain (AIDAC) is responsible for the surface-presentation of the α-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 °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 (β1) encompasses approximately 10 kDa to represent a completely surface-exposed segment while the C-terminal compact core domain (β2) remains integrated in the membrane as a β-barrel-like structure. Though the β2-core structure alone harbours all the information for the outer membrane integration of AIDAC it is additionally stabilized by the β1-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 β2-core in black-lipid membranes could not be demonstrated.

AIDA autotransporter β-barrel circular dichroism epitope tagging heat modifiability membrane translocator renaturation 

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Copyright information

© Kluwer Academic Publishers 2001

Authors and Affiliations

  • Marc P. J. Konieczny
    • 1
  • Inga Benz
    • 1
  • Britta Hollinderbäumer
    • 1
  • Christina Beinke
    • 1
  • Michael Niederweis
    • 2
    Email author
  • M. Alexander Schmidt
    • 1
  1. 1.Institut für Infektiologie, Zentrum für Molekularbiologie der Entzündung (ZMBE)Westfälische Wilhelms-Universität MünsterGermany
  2. 2.Lehrstuhl für MikrobiologieFriedrich-Alexander-Universität Erlangen-NürnbergErlangenGermany (Author for correspondence; E-mail

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