title: The periplasmic cavity of LacY mutant Cys154→Gly: how open is open?
abstract: The lactose permease from Escherichia coli (LacY) is a galactoside/H(+) symporter that catalyzes the coupled stoichiometric transport of a sugar and an H(+) across the cytoplasmic membrane. X-ray crystal structures of WT LacY and the conformationally restricted mutant Cys154→Gly exhibit an inward-facing conformation with a tightly sealed periplasmic side and a deep central cleft or cavity open to the cytoplasm. Although the crystal structures may give the impression that LacY is a rigid molecule, multiple converging lines of evidence demonstrate that galactoside binding to WT LacY induces reciprocal opening and closing of periplasmic and cytoplasmic cavities, respectively. By this means, the sugar- and H(+)-binding sites in the middle of the molecule are exposed alternatively to either side of the membrane. In contrast to the crystal structure, biochemical/biophysical studies with mutant Cys154→Gly show that the periplasmic side is paralyzed in an open-outward conformation. In this study, a rigid, funnel-shaped, maleimide-containing molecule was used to probe the periplasmic cavity of a pseudo-WT and the Cys154→Gly mutant by site-directed alkylation. The findings provide strong support for previous observations and indicate further that the external opening of the periplasmic cleft in the mutant is patent to the extent of at least 8.5 Å in the absence of sugar or about half that of the WT cavity with bound galactoside.
authors: Department of Physiology and ‡Department of Microbiology, Immunology & Molecular Genertics, §Molecular Biology Institute, University of California Los Angeles , Los Angeles, California 90095, United States.
authors: Xiaoxu
authors: X
authors: Jiang
authors: Arnold J M
authors: AJ
authors: Driessen
authors: Ben L
authors: BL
authors: Feringa
authors: H Ronald
authors: HR
authors: Kaback
mesh_terms: D001665:Binding Sites; D002462:Cell Membrane / Q000378:metabolism; D018360:Crystallography, X-Ray; D003593:Cytoplasm / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism; D005690:Galactose / Q000378:metabolism; D008301:Maleimides / Q000737:chemistry; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism / Q000648:ultrastructure; D019897:Periplasm / Q000737:chemistry / Q000378:metabolism*; D011487:Protein Conformation; D014320:Triphenylmethyl Compounds / Q000737:chemistry
chemical_list: C584312:1-(4-tritylphenyl)pyrrole-2,5-dione; D029968:Escherichia coli Proteins; D008301:Maleimides; D026901:Membrane Transport Proteins; D014320:Triphenylmethyl Compounds; C021499:lactose permease; D005690:Galactose
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