Modular organization of the AIDA autotransporter translocator:: The N-terminal β1-domain is surface-exposed and stabilizes the transmembrane β2-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 (AIDA C) 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 degreesC. Reporter-epitope insertions were used to analyze the location of regions of membrane-integrated AIDA C relative to the membrane. The modified topological model developed for the AIDA translocator suggests the N-terminal domain (beta (1)) encompasses approximately 10 kDa to represent a completely surface-exposed segment while the C-terminal compact core domain (beta (2)) remains integrated in the membrane as a beta -barrel-like structure. Though the beta (2)-core structure alone harbours all the information for the outer membrane integration of AIDA C it is additionally stabilized by the beta (1)-domain. Access to large amounts of complete as well as truncated AIDA C proteins facilitated the study of protein folding by CD and fluorescence spectroscopy. A potential pore forming activity of the translocator using the completely refolded AIDA C or the beta (2)-core in black-lipid membranes could not be demonstrated.