title: An Asymmetric Conformational Change in LacY.
abstract: Galactoside/H+symport by the lactose permease of Escherichia coli (LacY) involves reciprocal opening and closing of periplasmic and cytoplasmic cavities so that sugar- and H+-binding sites become alternatively accessible to either side of the membrane. After reconstitution into proteoliposomes, LacY with the periplasmic cavity sealed by cross-linking paired-Cys residues does not bind sugar from the periplasmic side. However, reduction of the S-S bond restores opening of the periplasmic cavity and galactoside binding. Furthermore, nanobodies that stabilize the double-Cys mutant in a periplasmic-open conformation and allow free access of galactoside to the binding site do so only after reduction of the S-S bond. In contrast, when cross-linked LacY is solubilized in detergent, galactoside binding is observed, indicating that the cytoplasmic cavity is patent. Sugar binding from the cytoplasmic side exhibits nonlinear stopped-flow kinetics, and analysis reveals a two-step process in which a conformational change precedes binding. Because the cytoplasmic cavity is spontaneously closing and opening in the symporter with a sealed periplasmic cavity, it is apparent that an asymmetrical conformational transition controls access of sugar to the binding site.
authors.affiliation: Department of Physiology, ‡Department of Microbiology, Immunology & Molecular Genetics, and §Molecular Biology Institute, University of California Los Angeles , Los Angeles, California 90095-7327, United States.
authors.forename: Irina
authors.initials: I
authors.lastname: Smirnova
authors.affiliation: Department of Physiology, ‡Department of Microbiology, Immunology & Molecular Genetics, and §Molecular Biology Institute, University of California Los Angeles , Los Angeles, California 90095-7327, United States.
authors.forename: Vladimir
authors.initials: V
authors.lastname: Kasho
authors.affiliation: Department of Physiology, ‡Department of Microbiology, Immunology & Molecular Genetics, and §Molecular Biology Institute, University of California Los Angeles , Los Angeles, California 90095-7327, United States.
authors.forename: Xiaoxu
authors.initials: X
authors.lastname: Jiang
authors.affiliation: Department of Physiology, ‡Department of Microbiology, Immunology & Molecular Genetics, and §Molecular Biology Institute, University of California Los Angeles , Los Angeles, California 90095-7327, United States.
authors.forename: H Ronald
authors.identifier: 0000-0003-0683-5810
authors.initials: HR
authors.lastname: Kaback
mesh_terms: D001665:Binding Sites; D001692:Biological Transport; D003545:Cysteine / Q000737:chemistry*; D004220:Disulfides / Q000737:chemistry*; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism; D005690:Galactose / Q000737:chemistry* / Q000378:metabolism; D015870:Gene Expression; D007700:Kinetics; D008958:Models, Molecular; D009004:Monosaccharide Transport Proteins / Q000737:chemistry* / Q000378:metabolism; D010084:Oxidation-Reduction; D011485:Protein Binding; D000072417:Protein Domains; D017433:Protein Structure, Secondary; D011510:Proteolipids / Q000737:chemistry* / Q000378:metabolism; D011522:Protons; D027981:Symporters / Q000737:chemistry* / Q000378:metabolism; D013816:Thermodynamics
chemical_list: D004220:Disulfides; D029968:Escherichia coli Proteins; C050221:LacY protein, E coli; D009004:Monosaccharide Transport Proteins; D011510:Proteolipids; D011522:Protons; D027981:Symporters; C020485:proteoliposomes; D003545:Cysteine; D005690:Galactose