Journal
EMBO JOURNAL
Volume 33, Issue 16, Pages 1831-1844Publisher
WILEY-BLACKWELL
DOI: 10.15252/embj.201387557
Keywords
five helix inverted repeat superfamily; hydantoin; membrane transport; Mhp1; molecular recognition; nucleobase-cation-symport; NCS1; family
Categories
Funding
- European Community [HEALTH-F4-2007-201924]
- European Drug Initiative for Channels and Transporters (EDICT) Consortium
- BBSRC [BB/C51725, BB/G023425/1]
- Leverhulme Trust
- Wellcome Trust at the Diamond Light Source [WT089809]
- BBSRC
- EPSRC
- Wellcome Trust
- Biotechnology and Biological Sciences Research Council [BEP17032, BB/C51725X/1, BB/H000267/1, BB/L002558/1, BBS/B/16011, BB/I019855/1, BB/G020043/1, B19456, BB/G023425/1] Funding Source: researchfish
- Engineering and Physical Sciences Research Council [EP/J010421/1, EP/K039202/1] Funding Source: researchfish
- BBSRC [BB/L002558/1, BB/G023425/1, BB/I019855/1, BB/G020043/1, BB/H000267/1] Funding Source: UKRI
- EPSRC [EP/K039202/1, EP/J010421/1] Funding Source: UKRI
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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.
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