title: Binding regions of outer membrane protein A in complexes with the periplasmic chaperone Skp. A site-directed fluorescence study.
abstract: 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 beta(x)W(n)-OmpA mutants, the tryptophan was located in beta-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 beta-barrel transmembrane domain (TMD) of OmpA. PDW(286)-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 beta(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 PDW(286)-OmpA, demonstrating binding of Skp to the entire beta-barrel domain but not to the PD of OmpA. Skp bound the TMD of OmpA asymmetrically, displaying much stronger interactions with strands beta(1) to beta(3) in the N-terminus than with strands beta(5) to beta(7) 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 beta-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.
authors.affiliation: Fachbereich Biologie, Universität Konstanz, Germany.
authors.forename: Jian
authors.initials: J
authors.lastname: Qu
authors.forename: Susanne
authors.initials: S
authors.lastname: Behrens-Kneip
authors.forename: Otto
authors.initials: O
authors.lastname: Holst
authors.forename: Jörg H
authors.initials: JH
authors.lastname: Kleinschmidt
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D003545:Cysteine / Q000235:genetics; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D008070:Lipopolysaccharides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D018832:Molecular Chaperones / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D046912:Multiprotein Complexes / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D016297:Mutagenesis, Site-Directed / Q000379:methods; D033902:Periplasmic Binding Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D011489:Protein Denaturation / Q000235:genetics; D017510:Protein Folding; D017434:Protein Structure, Tertiary / Q000235:genetics; D013050:Spectrometry, Fluorescence / Q000379:methods; D014364:Tryptophan / Q000235:genetics / Q000378:metabolism
chemical_list: D001425:Bacterial Outer Membrane Proteins; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D008070:Lipopolysaccharides; D018832:Molecular Chaperones; D046912:Multiprotein Complexes; D033902:Periplasmic Binding Proteins; C072721:Skp protein, E coli; C078167:OMPA outer membrane proteins; D014364:Tryptophan; D003545:Cysteine