Misfolding of a bacterial autotransporter

Abstract

The adhesin involved in diffuse adherence (AIDA) is an autotransporter protein that confers the diffuse adherence phenotype to certain diarrheagenic Escherichia coli strains. It consists of a 49 amino acid signal peptide, a 797 amino acid passenger domain, and a 440 amino acid b-domain integrated into the outer membrane. The b-domain consists of two parts: the b1-domain, which is predicted to form two b-strands on the bacterial cell surface, and the b2-domain, which constitutes the transmembrane domain. We have previously shown that the b-domain can be folded from the urea-denatured state when bound to a nickel column during purification. It has not been possible to achieve proper refolding of the b-domain in solution; instead, a misfolded state C is formed. Here, we characterize this misfolded state in greater detail, showing that despite being misfolded, C can be analyzed as a conventional conformational state, with cooperative unfolding in urea and SDS as well as showing simple exponential kinetics during its formation in the presence of lipid vesicles and detergent micelles. The kinetics of formation of C is sensitive to the lipid composition in vesicles. We have also attempted to identify biological factors that might aid folding of the b-domain to the properly folded state. However, no purified periplasmic or cytosolic chaperone was found to increase folding yields, and no factor in a periplasmic extract was identified that could bind to C. We conclude that it is the exposure to the unique spatial arrangement of the bacterial cell that leads to proper refolding of the b-domain.