Binding Regions of Outer Membrane Protein A in Complexes with the Periplasmic Chaperone Skp. A Site-Directed Fluorescence Study

Periplasmic Skp facilitates folding and membrane insertion of many outer membrane proteins (OMPs) into the outer membrane of Gram-negative bacteria. We have examined the binding sites of outer membrane protein A (OmpA) from Escherichia coli in its complexes with the membrane protein chaperone Skp and with Skp and lipopolysaccharide (LPS) by site-directed fluorescence spectroscopy. Single-Trp OmpA mutants, W n -OmpA, with tryptophan at position n in the polypeptide chain were isolated in the unfolded form in 8 M urea. In five β x W n -OmpA mutants, the tryptophan was located in β-strand x, in four l y W n -OmpA mutants, in outer loop y, and in three t z W n -OmpA mutants in turn z of the β-barrel transmembrane domain (TMD) of OmpA. PDW286-OmpA contained tryptophan in the periplasmic domain (PD). After dilution of the denaturant urea in aqueous solution, spectra indicated a more hydrophobic environment of the tryptophans in β x W n mutants in comparison to l y W n -OmpA and t z W n -OmpA, indicating that the loops and turns form the surface of hydrophobically collapsed OmpA, while the strand regions are less exposed to water. Addition of Skp increased the fluorescence of all OmpA mutants except PDW286-OmpA, demonstrating binding of Skp to the entire β-barrel domain but not to the PD of OmpA. Skp bound the TMD of OmpA asymmetrically, displaying much stronger interactions with strands β1 to β3 in the N-terminus than with strands β5 to β7 in the C-terminus. This asymmetry was not observed for the outer loops and the periplasmic turns of the TMD of OmpA. The fluorescence results demonstrated that all turns and loops l 1, l 2, and l 4 were as strongly bound to Skp as the N-terminal β-strands. Addition of five negatively charged LPS per one preformed Skp·W n -OmpA complex released the C-terminal loops l 2, l 3, and l 4 of the TMD of OmpA from the complex, while its periplasmic turn regions remained bound to Skp. Our results demonstrate that interactions of Skp·OmpA complexes with LPS change the conformation of OmpA in the Skp complex for facilitated insertion and folding into membranes.