title: Crystal structure of a LacY-nanobody complex in a periplasmic-open conformation.
abstract: The lactose permease of Escherichia coli (LacY), a dynamic polytopic membrane protein, catalyzes galactoside-H+symport and operates by an alternating access mechanism that exhibits multiple conformations, the distribution of which is altered by sugar binding. We have developed single-domain camelid nanobodies (Nbs) against a mutant in an outward (periplasmic)-open conformation to stabilize this state of the protein. Here we describe an X-ray crystal structure of a complex between a double-Trp mutant (Gly46→Trp/Gly262→Trp) and an Nb in which free access to the sugar-binding site from the periplasmic cavity is observed. The structure confirms biochemical data indicating that the Nb binds stoichiometrically with nanomolar affinity to the periplasmic face of LacY primarily to the C-terminal six-helix bundle. The structure is novel because the pathway to the sugar-binding site is constricted and the central cavity containing the galactoside-binding site is empty. Although Phe27 narrows the periplasmic cavity, sugar is freely accessible to the binding site. Remarkably, the side chains directly involved in binding galactosides remain in the same position in the absence or presence of bound sugar.
authors: State Key Laboratory of Membrane Biology, Center for Structural Biology, School of Life Sciences and School of Medicine, Tsinghua University, Beijing 100084, China.
authors: Xin
authors: X
authors: Jiang
authors: Department of Physiology, University of California, Los Angeles, CA 90095.
authors: Irina
authors: I
authors: Smirnova
authors: Department of Physiology, University of California, Los Angeles, CA 90095.
authors: Vladimir
authors: V
authors: Kasho
authors: State Key Laboratory of Membrane Biology, Center for Structural Biology, School of Life Sciences and School of Medicine, Tsinghua University, Beijing 100084, China.
authors: Jianping
authors: J
authors: Wu
authors: Advanced Photon Technology Division, Research Infrastructure Group, SR Life Science Instrumentation Unit, RIKEN/SPring-8 Center, Hyogo 679-5148, Japan.|Japan Science and Technology Agency, Precursory Research for Embryonic Science and Technology (PRESTO), 4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan.
authors: Kunio
authors: K
authors: Hirata
authors: State Key Laboratory of Membrane Biology, Center for Structural Biology, School of Life Sciences and School of Medicine, Tsinghua University, Beijing 100084, China.
authors: Meng
authors: M
authors: Ke
authors: VIB Center for Structural Biology Research, VIB, 1050 Brussels, Belgium.|Structural Biology Brussels, Vrije Universiteit Brussel, 1050 Brussels, Belgium.
authors: Els
authors: E
authors: Pardon
authors: VIB Center for Structural Biology Research, VIB, 1050 Brussels, Belgium.|Structural Biology Brussels, Vrije Universiteit Brussel, 1050 Brussels, Belgium.
authors: Jan
authors: J
authors: Steyaert
authors: State Key Laboratory of Membrane Biology, Center for Structural Biology, School of Life Sciences and School of Medicine, Tsinghua University, Beijing 100084, China.
authors: Nieng
authors: N
authors: Yan
authors: Department of Physiology, University of California, Los Angeles, CA 90095; rkaback@mednet.ucla.edu.|Department of Microbiology, Immunology, and Molecular Genetics, University of California, Los Angeles, CA 90095.|Molecular Biology Institute, University of California, Los Angeles, CA 90095.
authors: H Ronald
authors: HR
authors: Kaback
mesh_terms: D001665:Binding Sites; D018360:Crystallography, X-Ray; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D008958:Models, Molecular; D009004:Monosaccharide Transport Proteins / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D009154:Mutation; D019897:Periplasm / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D061905:Single-Domain Antibodies / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism; D027981:Symporters / Q000737:chemistry* / Q000235:genetics / Q000276:immunology
chemical_list: D029968:Escherichia coli Proteins; C050221:LacY protein, E coli; D009004:Monosaccharide Transport Proteins; D061905:Single-Domain Antibodies; D027981:Symporters
keywords: X-ray structure; bioenergetics; fluorescence; kinetics; membrane transporter