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Antonie Van Leeuwenhoek. 2001 Oct;80(1):19-34.

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

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1
Institut für Infektiologie, Zentrum für Molekularbiologie der Entzündung, Westfälische Wilhelms-Universität Münster, Germany.

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.

PMID:
11761364
[Indexed for MEDLINE]
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