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Molecular mechanism of ligand recognition by membrane transport protein, Mhp1

DOI: 10.15252/embj.201387557 DOI Help
PMID: 24952894 PMID Help

Authors: Katie J Simmons (School of Chemistry and Astbury Centre for Structural Molecular Biology, University of Leeds) , Scott Jackson (School of Chemistry and Astbury Centre for Structural Molecular Biology, University of Leeds) , Florian Brueckner (Membrane Protein Laboratory, Diamond Light Source) , Simon Patching (School of Biomedical Sciences and Astbury Centre for Structural Molecular Biology, University of Leeds) , O. Beckstein (Department of Physics, Arizona State University) , Ekaterina Ivanova (School of Biomedical Sciences and Astbury Centre for Structural Molecular Biology, University of Leeds) , Tian Geng (Membrane Protein Laboratory, Diamond Light Source) , Simone Weyand (Membrane Protein Laboratory, Diamond Light Source) , David Drew (Division of Molecular Biosciences, Membrane Protein Crystallography Group, Imperial College) , J. Lanigan (School of Chemistry and Astbury Centre for Structural Molecular Biology, University of Leeds) , D. J. Sharples (School of Biomedical Sciences and Astbury Centre for Structural Molecular Biology, University of Leeds) , M. S. Sansom (Department of Biochemistry) , So Iwata (Diamond Light Source) , C. W. Fishwick (School of Chemistry and Astbury Centre for Structural Molecular Biology, University of Leeds) , A. P. Johnson (School of Chemistry and Astbury Centre for Structural Molecular Biology, University of Leeds) , Alex Cameron (Membrane Protein Laboratory, Diamond Light Source) , Peter Henderson (School of Biomedical Sciences and Astbury Centre for Structural Molecular Biology, University of Leeds)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: The Embo Journal , VOL 33 (16) , PAGES 1831 - 1844

State: Published (Approved)
Published: August 2014

Open Access Open Access

Abstract: The hydantoin transporter Mhp1 is a sodium-coupled secondary active transport protein of the nucleobase-cation-symport family and a member of the widespread 5-helix inverted repeat superfamily of transporters. The structure of Mhp1 was previously solved in three different conformations providing insight into the molecular basis of the alternating access mechanism. Here, we elucidate detailed events of substrate binding, through a combination of crystallography, molecular dynamics, site-directed mutagenesis, biochemical/biophysical assays, and the design and synthesis of novel ligands. We show precisely where 5-substituted hydantoin substrates bind in an extended configuration at the interface of the bundle and hash domains. They are recognised through hydrogen bonds to the hydantoin moiety and the complementarity of the 5-substituent for a hydrophobic pocket in the protein. Furthermore, we describe a novel structure of an intermediate state of the protein with the external thin gate locked open by an inhibitor, 5-(2-naphthylmethyl)-L-hydantoin, which becomes a substrate when leucine 363 is changed to an alanine. We deduce the molecular events that underlie acquisition and transport of a ligand by Mhp1.

Journal Keywords: Binding; Biological; Crystallography; X-Ray; Hydantoins; Hydrogen; Ligands; Micrococcaceae; Models; Molecular; Mutagenesis; Site-Directed; Mutation; Protein; Structure-Activity Relationship

Subject Areas: Biology and Bio-materials

Diamond Offline Facilities: Membrane Protein Laboratory (MPL)
Instruments: I02-Macromolecular Crystallography , I03-Macromolecular Crystallography , I04-Macromolecular Crystallography

Documents:
embj201387557.pdf