Crystallization of the hydantoin transporter Mhp1 from<i>Microbacterium liquefaciens</i>

Shimamura, Tatsuro; Yajima, Seishi; Suzuki, Shunichi; Rutherford, Nicholas G.; O’Reilly, John; Henderson, Peter J. F.; Iwata, So

doi:10.1107/s1744309108036920

Cited by 12 publications

(10 citation statements)

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“…Crystals of IMH‐Mhp1, BH‐Mhp1, BVH‐Mhp1, NMH‐Mhp1 were grown essentially as previously described (Shimamura et al , , ; Weyand et al , ). Details of crystallisation, data collection and structure refinement are in Supplementary Methods.…”

Section: Methodsmentioning

confidence: 99%

Molecular mechanism of ligand recognition by membrane transport protein, Mhp1

et al. 2014

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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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Section: Methodsmentioning

confidence: 99%

Molecular mechanism of ligand recognition by membrane transport protein, Mhp1

et al. 2014

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“…Here, we present the substrate-free inward-facing structure of Mhp1, a sodium-hydantoin transporter from Microbacterium liquefaciens (9, 22, 23) at 3.8 Å resolution (Fig. 1).…”

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Molecular Basis of Alternating Access Membrane Transport by the Sodium-Hydantoin Transporter Mhp1

Shimamura

Weyand

Beckstein

et al. 2010

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The structure of the sodium-benzylhydantoin transport protein Mhp1 from Microbacterium liquefaciens comprises a five-helix inverted repeat, which is widespread among secondary transporters. Here, we report the crystal structure of an inward-facing conformation of Mhp1 at 3.8 angstroms resolution, complementing its previously described structures in outward-facing and occluded states. From analyses of the three structures and molecular dynamics simulations, we propose a mechanism for the transport cycle in Mhp1. Switching from the outward- to the inward-facing state, to effect the inward release of sodium and benzylhydantoin, is primarily achieved by a rigid body movement of transmembrane helices 3, 4, 8, and 9 relative to the rest of the protein. This forms the basis of an alternating access mechanism applicable to many transporters of this emerging superfamily.

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“…The substrates for Mhp1are hydantoins substituted with aromatic groups at the 5-position. The wild-type protein contains 489 amino acids (Suzuki and Henderson 2006), modestly modified in a genetic construct at the N-terminus and C-terminus, where a (His) 6 tag is incorporated to facilitate amplified expression, purification, and crystallization (Suzuki and Henderson 2006;Shimamura et al 2008). The transport reaction of Mhp1 is:…”

Section: Definitionmentioning

confidence: 99%