Molecular mechanism of ligand recognition by membrane transport protein, Mhp1

Katie J. Simmons, Scott M. Jackson, Florian Brueckner, Simon G. Patching, Oliver Beckstein, Ekaterina Ivanova, Tian Geng, Simone Weyand, David Drew, Joseph Lanigan, David J. Sharples, Mark S P Sansom, So Iwata, Colin W G Fishwick, A. Peter Johnson, Alexander D. Cameron, Peter J F Henderson

Research output: Contribution to journalArticlepeer-review

39 Scopus citations

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. Synopsis Structure-function and molecular dynamics analysis of the hydantoin active transporter Mhp1 yields a novel intermediate state and delineates the basis for substrate specificity and membrane transport. Hydantoin substrates like indolylmethylhydantoin (IMH) bind to the LeuT-like Mhp1 transporter in an extended conformation Selectivity of Mhp1 for the substrate is conferred by hydrogen bonds to the hydantoin moiety and the fit of aromatic substituent into a hydrophobic pocket Naphthylmethylhydantoin (NMH) inhibits Mhp1 but is not transported Crystal structure of Mhp1 with NMH shows TMH10 to adopt the position seen in the outward-open rather than the occluded state. Mutation of Leu363Ala in TMH10 of Mhp1 converts NMH from an inhibitor to a substrate. Structure-function and molecular dynamics analysis of the hydantoin active transporter Mhp1 yields a novel intermediate state and delineates the basis for substrate specificity and membrane transport.

Original languageEnglish (US)
Pages (from-to)1831-1844
Number of pages14
JournalEMBO Journal
Volume33
Issue number16
DOIs
StatePublished - Aug 18 2014

Keywords

  • Mhp1
  • five helix inverted repeat superfamily
  • hydantoin
  • membrane transport
  • molecular recognition
  • nucleobase-cation-symport, NCS1, family

ASJC Scopus subject areas

  • Neuroscience(all)
  • Molecular Biology
  • Biochemistry, Genetics and Molecular Biology(all)
  • Immunology and Microbiology(all)

Fingerprint Dive into the research topics of 'Molecular mechanism of ligand recognition by membrane transport protein, Mhp1'. Together they form a unique fingerprint.

Cite this