Molecular mechanism of ligand recognition by membrane transport protein, Mhp1

• Katie J Simmons ^[1] ; Scott M Jackson ^[1] ; Florian Brueckner ^[5] ; Simon G Patching ^[1] ; Oliver Beckstein ^[2] ; Ekaterina Ivanova ^[1] ; Tian Geng ^[5] ; Simone Weyand ^[5] ; David Drew ^[3] ; Joseph Lanigan ^[1] ; David J Sharples ^[1] ; Mark SP Sansom ^[4] ; So Iwata ^[5] ; Colin WG Fishwick ^[1] ; A Peter Johnson ^[1] ; Alexander D Cameron ^[6] ; Peter JF Henderson ^[1]
  1. [1] University of Leeds

     University of Leeds

     Reino Unido

     
  2. [2] Arizona State University

     Arizona State University

     Estados Unidos

     
  3. [3] Imperial College London

     Imperial College London

     Reino Unido

     
  4. [4] University of Oxford

     University of Oxford

     Oxford District, Reino Unido

     
  5. [5] 3 Membrane Protein Laboratory, Diamond Light Source, Harwell Science and Innovation Campus Chilton, Didcot, UK; 4 Division of Molecular Biosciences, Membrane Protein Crystallography Group, Imperial College London, UK; 5 Rutherford Appleton Laboratory, Research Complex at Harwell Harwell, Oxford, Didcot, UK
  6. [6] 3 Membrane Protein Laboratory, Diamond Light Source, Harwell Science and Innovation Campus Chilton, Didcot, UK; 4 Division of Molecular Biosciences, Membrane Protein Crystallography Group, Imperial College London, UK; 8 School of Life Sciences, University of Warwick Coventry, UK

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• Localización: EMBO journal: European Molecular Biology Organization, ISSN 0261-4189, Vol. 33, Nº. 16, 2014, págs. 1831-1844
• Idioma: inglés
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• Resumen

  • 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.