title: Molecular mechanism of ligand recognition by membrane transport protein, Mhp1.
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.
authors: School of Chemistry and Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, UK.
authors: Katie J
authors: KJ
authors: Simmons
authors: School of Biomedical Sciences and Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, UK.
authors: Scott M
authors: SM
authors: Jackson
authors: Membrane Protein Laboratory, Diamond Light Source Harwell Science and Innovation Campus, Chilton, Didcot, UK Division of Molecular Biosciences, Membrane Protein Crystallography Group Imperial College, London, UK Rutherford Appleton Laboratory, Research Complex at Harwell, Harwell, Oxford, Didcot, UK.
authors: Florian
authors: F
authors: Brueckner
authors: School of Biomedical Sciences and Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, UK.
authors: Simon G
authors: SG
authors: Patching
authors: Department of Physics, Arizona State University, Tempe, AZ, USA Department of Biochemistry, University of Oxford, Oxford, UK.
authors: Oliver
authors: O
authors: Beckstein
authors: School of Biomedical Sciences and Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, UK.
authors: Ekaterina
authors: E
authors: Ivanova
authors: Membrane Protein Laboratory, Diamond Light Source Harwell Science and Innovation Campus, Chilton, Didcot, UK Division of Molecular Biosciences, Membrane Protein Crystallography Group Imperial College, London, UK Rutherford Appleton Laboratory, Research Complex at Harwell, Harwell, Oxford, Didcot, UK.
authors: Tian
authors: T
authors: Geng
authors: Membrane Protein Laboratory, Diamond Light Source Harwell Science and Innovation Campus, Chilton, Didcot, UK Division of Molecular Biosciences, Membrane Protein Crystallography Group Imperial College, London, UK Rutherford Appleton Laboratory, Research Complex at Harwell, Harwell, Oxford, Didcot, UK.
authors: Simone
authors: S
authors: Weyand
authors: Division of Molecular Biosciences, Membrane Protein Crystallography Group Imperial College, London, UK.
authors: David
authors: D
authors: Drew
authors: School of Chemistry and Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, UK.
authors: Joseph
authors: J
authors: Lanigan
authors: School of Biomedical Sciences and Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, UK.
authors: David J
authors: DJ
authors: Sharples
authors: Department of Biochemistry, University of Oxford, Oxford, UK.
authors: Mark S P
authors: MS
authors: Sansom
authors: Membrane Protein Laboratory, Diamond Light Source Harwell Science and Innovation Campus, Chilton, Didcot, UK Division of Molecular Biosciences, Membrane Protein Crystallography Group Imperial College, London, UK Rutherford Appleton Laboratory, Research Complex at Harwell, Harwell, Oxford, Didcot, UK.
authors: So
authors: S
authors: Iwata
authors: School of Chemistry and Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, UK p.j.f.henderson@leeds.ac.uk a.cameron@warwick.ac.uk c.w.g.fishwick@leeds.ac.uk.
authors: Colin W G
authors: CW
authors: Fishwick
authors: School of Chemistry and Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, UK.
authors: A Peter
authors: AP
authors: Johnson
authors: Membrane Protein Laboratory, Diamond Light Source Harwell Science and Innovation Campus, Chilton, Didcot, UK Division of Molecular Biosciences, Membrane Protein Crystallography Group Imperial College, London, UK School of Life Sciences, University of Warwick, Coventry, UK p.j.f.henderson@leeds.ac.uk a.cameron@warwick.ac.uk c.w.g.fishwick@leeds.ac.uk.
authors: Alexander D
authors: AD
authors: Cameron
authors: School of Biomedical Sciences and Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, UK p.j.f.henderson@leeds.ac.uk a.cameron@warwick.ac.uk c.w.g.fishwick@leeds.ac.uk.
authors: Peter J F
authors: PJ
authors: Henderson
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D001692:Biological Transport; D018360:Crystallography, X-Ray; D006827:Hydantoins / Q000378:metabolism; D006860:Hydrogen Bonding; D008024:Ligands; D008835:Micrococcaceae / Q000737:chemistry; D008958:Models, Molecular; D056004:Molecular Dynamics Simulation; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011487:Protein Conformation; D013329:Structure-Activity Relationship
chemical_list: D001426:Bacterial Proteins; D006827:Hydantoins; D008024:Ligands; C106150:Mhp1 protein, Mycoplasma; C067244:5-indolylmethylhydantoin; C045467:hydantoin 454C77
keywords: Mhp1; five helix inverted repeat superfamily; hydantoin; membrane transport; molecular recognition; nucleobase‐cation‐symport, NCS1, family