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.

Keywords: Mhp1; five helix inverted repeat superfamily; hydantoin; membrane transport; molecular recognition; nucleobase‐cation‐symport, NCS1, family.

Figure 1. Binding of substrates in Mhp1

A, B Superposition of the outward-open structure (PDB code 2JLN) onto the IMH-bound structure, optimised using the bundle helices. The IMH structure is shown with the bundle in red, the hash motif in yellow, TMHs 5 and 10 in blue and the C-terminal helices in magenta. The outward-open structure is shown in grey. The L-IMH (green spheres) and sodium ion (magenta) bind between the hash and bundle motifs. (A) shows an overview of all helices and (B) a close up. The arrows show the main conformational changes that occur upon L-IMH binding. Arrow A: the hash motif rotates towards the bundle with the C-terminal helices partially following. Arrows B and C: Trp117 and Trp220 rotate towards the hydantoin moiety and the 5-indole substituent, respectively, of L-IMH. Arrow D: TMH10 flexes and packs over the IMH. C The extended form of L-IMH in the binding site illustrated using the SPROUT format (Materials and Methods and Supplementary Methods) to show the indole moiety in a hydrophobic pocket (green). D Schematic of interactions made between L-IMH and the protein. Possible hydrogen bonds are indicated by straight dashed lines and hydrophobic interactions by curved dashed lines. E The extended form of L-BH is oriented similarly to L-IMH with its benzyl moiety in the hydrophobic pocket. F Schematic of the interactions made by L-BH with Mhp1. Data information: In (C and E) green represents regions where a hydrophobic interaction can be made, blue represents regions containing hydrogen-bond donor atoms, and red represents regions containing hydrogen-bond acceptor atoms.

Figure 2. Molecular dynamics simulations of Mhp1 and its ligands

A–C Molecular conformations and conformational free energy landscape of L-BH, L-IMH and L-NMH in aqueous solution suggested by molecular dynamics simulations L-BH (A), L-IMH (B) and L-NMH (C) in solution: The conformation of the hydantoin derivatives are described by the dihedral angles χ¹ and χ² as indicated in the insets. The most probable conformations are indicated by the minima in the free energy of the system (in kT) as a function of the dihedral angles. Regions not sampled by the equilibrium simulations are white; other possible minima would be separated by barriers larger than 6 kT from the accessible regions. D–F Hydrogen bonds between substrates and Mhp1 as seen in MD simulations. The ligand and important residues are shown as sticks with hydrogen bonds as broken black lines. Helices TM1 and TM6 from the bundle are in red and TM3 and TM8 from the hash motif in yellow (parts of TM3 were removed for clarity); a sodium ion in the Na2 site is visible in the background. Water density is shown as a cyan mesh, contoured at 1.5 times the bulk value. Equivalent atoms on the ligands are labelled. (D) L-BH [from simulation 5FH(g⁺)MD_002]. (E) L-IMH [from simulation IMH(g^–)MD_002]. (F) Clustered fingerprint analysis of hydrogen bonds. The occupancy (average number of hydrogen bonds between ligand atoms and protein or solvent atoms) from all MD simulations was clustered to show the most commonly occurring hydrogen bonding patterns. Rows describe individual hydrogen bonds (identified by donor and acceptor heavy atom) while columns label individual simulations; hydrogen bonds labelled in red were also seen in the crystal structures and in docking while blue ones indicate bonds to water molecules present in the simulation. The ligand is denoted in the simulation name as either 5FH (L-BH) or IMH (L-IMH) together with the starting conformation of the χ₁ dihedral angle and the simulation number within the set. Chemically equivalent ligand atoms are treated as the same in the analysis (indicated by the generic label “LIG” instead of “5FH” or “IMH”). Hydrogen-bonded water molecules are also treated as chemically equivalent (“SOL” for solvent).

Figure 3. Impairment of hydantoin uptake in mutants of Mhp1

The accumulation of ¹⁴C-L-IMH (50 μM initial external concentration) was measured for 15 s in cells expressing the wild-type or mutant Mhp1 proteins (Materials and Methods and Supplementary Methods). All measurements were normalised to percentages by comparison with the wild-type value of 0.57 ± 0.01 (s.e.m., n = 34) nmol/mg dry mass. Error bars represent the s.e.m. of at least triplicate measurements. All assays were performed in the presence of 150 mM NaCl.

Figure 4. Ligand specificity of Mhp1 determined by uptake assays

A, B Accumulation of ¹⁴C-L-IMH (50 μM initial external concentration) into wild-type cells was measured for 15 s (Materials and Methods and Supplementary Methods): (A) in the presence of 500 μM of the indicated unlabelled compound; and (B) dose–response data for ¹⁴C-L-IMH uptake in the presence of 0–500 μM of selected unlabelled compound. All measurements were normalised to percentages by comparison with the wild-type value of 0.57 ± 0.01 (s.e.m., n = 34) nmol/mg dry mass, and the error bars represent the s.e.m. of at least triplicate measurements. C Uptakes of the indicated radioisotope-labelled compounds (50 μM initial external concentration, Materials and Methods and Supplementary Methods), into wild-type cells were measured for 15 s for the original Mhp1 protein and for the L363A mutant as indicated at least in triplicate on each of two cell preparations and the s.e.m. calculated for at least six assays. Hyd = hydantoin; All = allantoin. L-tryptophan and D/L allantoin were tested as controls in both (A) and (C).

Figure 5. Structure of wild-type Mhp1 with bound L-NMH

A Docking pose of L-NMH illustrated using SPROUT as for Fig1. B Comparison of the crystal structure of L-NMH (green) with the outward-open ligand-free structure (grey) and the complex with L-IMH (coloured as in Fig1). C, D Potential hydrogen bonding interactions between L-NMH and the protein as in Fig1.

Figure 6. Scheme for binding of ligands and transport by the Mhp1 protein

Upon binding to the outward-open conformation of Mhp1 (1) both the substrate IMH (right) and the inhibitor NMH (left) induce a number of conformational changes to the protein. The hash motif (yellow) moves towards the bundle (red), and Trp117 and Trp220 rotate to interact with the ligand (denoted by arrows A, B and C respectively). This results in a partial occlusion of the outward cavity, shown here by a solid line approximately defining the entrance to the cavity from the outside. A conformational change of TMH10 (D) results in the complete occlusion of the substrate in the binding site (3), and subsequently, the protein switches to the inward-facing form. For NMH (2*), TMH10 cannot adopt the position observed for NMH and transport does not occur. The scheme has been based on the crystal structures of states 1, 2* and 3.