Misfolding of a bacterial autotransporter.
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 beta-domain integrated into the outer membrane. The beta-domain consists of two parts: the beta(1)-domain, which is predicted to form two beta-strands on the bacterial cell surface, and the beta(2)-domain, which constitutes the transmembrane domain. We have previously shown that the beta-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 beta-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 beta-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 beta-domain.
Mogensen
Jesper E
JE
Department of Life Sciences, Aalborg University, Sohngaardsholmsvej 49, DK-9000 Aalborg, Denmark.
Kleinschmidt
Jörg H
JH
Schmidt
M Alexander
MA
Otzen
Daniel E
DE
D018830:Adhesins, Escherichia coli / Q000737:chemistry* / Q000378:metabolism; D000817:Anilino Naphthalenesulfonates / Q000378:metabolism; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism; D018834:Chaperonin 60 / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D003902:Detergents / Q000737:chemistry; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000378:metabolism; D006360:Heat-Shock Proteins / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008563:Membrane Lipids / Q000737:chemistry; D018832:Molecular Chaperones / Q000378:metabolism; D019696:Peptidylprolyl Isomerase / Q000378:metabolism; D033903:Periplasmic Proteins / Q000378:metabolism; D011489:Protein Denaturation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D012697:Serine Endopeptidases / Q000378:metabolism; D012995:Solubility; D014867:Water / Q000737:chemistry
C075559:AIDA-I protein, E coli; D018830:Adhesins, Escherichia coli; D000817:Anilino Naphthalenesulfonates; D002352:Carrier Proteins; D018834:Chaperonin 60; D004268:DNA-Binding Proteins; D003902:Detergents; D029968:Escherichia coli Proteins; D006360:Heat-Shock Proteins; D008563:Membrane Lipids; D018832:Molecular Chaperones; D033903:Periplasmic Proteins; D014867:Water; C072721:Skp protein, E coli; C027132:1-anilino-8-naphthalenesulfonate; C061682:DegP protease; D012697:Serine Endopeptidases; C490900:SurA protein, E coli; D019696:Peptidylprolyl Isomerase