Modular organization of the AIDA autotransporter translocator: the N-terminal beta1-domain is surface-exposed and stabilizes the transmembrane beta2-domain.
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.
Konieczny MPJ
Institut für Infektiologie, Zentrum für Molekularbiologie der Entzündung, Westfälische Wilhelms-Universität Münster, Germany.
Benz
I
I
Hollinderbäumer
B
B
Beinke
C
C
Niederweis
M
M
Schmidt
M A
MA
D018830:Adhesins, Escherichia coli / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D002462:Cell Membrane / Q000378:metabolism*; D002942:Circular Dichroism; D010450:Endopeptidases / Q000378:metabolism; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006358:Hot Temperature; D008958:Models, Molecular; D008969:Molecular Sequence Data; D017510:Protein Folding; D011498:Protein Precursors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion; D013050:Spectrometry, Fluorescence
C075559:AIDA-I protein, E coli; D018830:Adhesins, Escherichia coli; D011498:Protein Precursors; D011993:Recombinant Fusion Proteins; D010450:Endopeptidases