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Biochem J
2015 Feb 15;4661:177-88. doi: 10.1042/BJ20141223.
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Identification of key binding site residues of MCT1 for AR-C155858 reveals the molecular basis of its isoform selectivity.
Nancolas B
,
Sessions RB
,
Halestrap AP
.
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The proton-linked monocarboxylate transporters (MCTs) are required for lactic acid transport into and out of all mammalian cells. Thus, they play an essential role in tumour cells that are usually highly glycolytic and are promising targets for anti-cancer drugs. AR-C155858 is a potent MCT1 inhibitor (Ki ~2 nM) that also inhibits MCT2 when associated with basigin but not MCT4. Previous work [Ovens, M.J. et al. (2010) Biochem. J. 425, 523-530] revealed that AR-C155858 binding to MCT1 occurs from the intracellular side and involves transmembrane helices (TMs) 7-10. In the present paper, we generate a molecular model of MCT4 based on our previous models of MCT1 and identify residues in the intracellular substrate-binding cavity that differ significantly between MCT4 and MCT1/MCT2 and so might account for differences in inhibitor binding. We tested their involvement using site-directed mutagenesis (SDM) of MCT1 to change residues individually or in combination with their MCT4 equivalent and determined inhibitor sensitivity following expression in Xenopus oocytes. Phe360 and Ser364 were identified as important for AR-C155858 binding with the F360Y/S364G mutant exhibiting >100-fold reduction in inhibitor sensitivity. To refine the binding site further, we used molecular dynamics (MD) simulations and additional SDM. This approach implicated six more residues whose involvement was confirmed by both transport studies and [3H]-AR-C155858 binding to oocyte membranes. Taken together, our data imply that Asn147, Arg306 and Ser364 are important for directing AR-C155858 to its final binding site which involves interaction of the inhibitor with Lys38, Asp302 and Phe360 (residues that also play key roles in the translocation cycle) and also Leu274 and Ser278.
Figure 1. Residue differences between MCT1 and MCT4 isoforms within TM helices 7–10(A) Cα atoms of MCT4 homology model (green) are super-imposed on to the model of MCT1 (blue). Residues lying in predicted TM helices 7–10 which differ between isoforms are coloured in red (MCT1) and their equivalent in MCT4 in orange and their positions indicated. (B) Example of a simulation set-up. The model of MCT1 (dark blue) is embedded in a POPC bilayer (purple) and solvated to a box size of 12 nm3 in water containing 0.15 M NaCl (light blue).
Figure 2. Docking of AR-C155858 to MCT1Docking to the inward (A) and intermediate (B) conformations of MCT1 before (i) and after (ii) simulation using GOLD. The positions of AR-C155858 are shown in stick form to demonstrate the change in pose over 160–200 ns.
Figure 3. Inhibition of the S364G/F360Y and S364A/F360Y mutantsInhibition of L-lactate transport by mutants expressed in Xenopus oocytes. Uptake was corrected for MCT-independent transport in water-injected oocytes (n=5) and data are presented as percentage inhibition ±% S.E.M. (error bars) for S364G/F360Y (A) and S364A/F360Y (B). For each inhibitor concentration the number of oocytes used was 15–64 (A) or 15–25 (B) with fewer oocytes used at the higher inhibitor concentrations (>500 nM) where variation between oocytes was less. Data for MCT4 are included in (A) for comparison. Note that where no error bars are shown they lie within the data symbol. Plasma membrane expression of the MCTs was determined by Western blotting of membrane fractions using the C-terminal MCT1 antibody.
Figure 4. Binding of [3H]-AR-C155858 to MCT1 and MCT1-mutants(A) Oocytes were injected with RNA or water 3 days prior to incubation of 20 oocytes in 3 ml of pH 6.0 transport buffer containing 50 nM AR-C155858 (specific activity 933 Bq/pmol [3H]-AR-C155858) for 45 min at room temperature. Membranes were then isolated and the pellet containing the membrane fraction solubilized in 20% SDS before scintillation counting. Non-specific binding was corrected for by subtracting the bound inhibitor for water-injected oocytes and data are shown as means±S.E.M. of 20–50 individual oocytes from 2–5 separate experiments. (B) Plasma membrane proteins (10 μg) were separated by SDS/PAGE and MCT1 expression detected by Western blot using the C-terminal MCT1 antibody. Equal loading was confirmed by Coomassie staining of the PVDF membrane. Black lines indicate separate blots.
Figure 5. Inward-intermediate conformation of MCT1 taken from a structure observed during the lactate simulation(A) Conformation of MCT1 after dynamic simulation for 200 ns with lactate present in the substrate channel. Lactate interacted with Arg306 and Lys38 during this simulation but has been removed from the resulting structure for simulation with inhibitor. Inset shows the distance between Lys38 and Asp302 at the end of the simulation. (B) Position of AR-C155858 after 200 ns simulation when docked initially to a 10 Å radius around Lys38. Lys38, Asp302, Phe360, Arg306 and Ser364 are shown. (C) RMSD of all AR-C155858 atoms versus or with respect to inward-intermediate simulation time.
Figure 6. The role of residues in close proximity to AR-C155858 during the inward-intermediate simulation in inhibitor binding(A) The position of residues Met65, Met69, Leu274 and Ser278 relative to AR-C155858 is shown after 200 ns simulation in the inward-intermediate conformation of MCT1. (B) The distance between AR-C155858 and residues Met65, Met69, Leu274 and Ser278 during the time course of the simulation. Distances shown are between atoms: M65S to AR-C–HG1; M69CE to AR-C–His121; L274HG to AR-C–SE1 and S278OG to AR-C–His103. (C) Inhibition of lactate uptake by M65L/M69L, L274P and S278V mutants in response to increasing concentrations of AR-C155858. (D) Plasma membrane expression of the MCT1 mutants shown by immunofluorescence microscopy and Western blot using the C-terminal MCT1 antibody.
Figure 7. Inhibition of a quadruple mutant of MCT1 by AR-C155858(A) Absolute rates of L-lactate uptake by oocytes expressing WT and the quadruple mutant of MCT1 (L274P/S278V/F360Y/S364G) at increasing concentrations of AR-C155858. Data are presented as mean±S.E.M (error bars) of 5–37 separate oocytes. Fewer oocytes were used for higher AR-C155858 concentrations due to the smaller variation in lactate uptake observed. All data are corrected for uptake by water-injected oocytes (n=5). Note that where no error bars are shown, they lie within the data symbol. (B) The same data are expressed for the percentage inhibition of transport and additional data are shown for the double mutant S364G/F360Y and for WT MCT4. (C) Plasma membrane expression of the MCTs is shown by Western blotting using the C-terminal MCT1 antibody.
Figure 8. Position of AR-C155858 after 167–200 ns simulation in the WT MCT1 or S364G/F360Y mutantS-AR-C155858 was docked into the inward-intermediate conformation of MCT1 and the best scoring solution taken for simulation (167–200 ns). The same initial position and conformation of AR-C155858 was used as the start for each simulation shown. (A) The position of S-AR-C155858 after 167 ns simulation in the WT MCT1 model. The hydrogen bond between Asp302 and AR-C155858 hydroxy group is indicated (1.67 Å). (B) The position of S-AR-C155858 after simulation in a model containing the mutations S364G and F360Y (indicated). (C) The position of R-AR-C155858 after simulation in the WT MCT1 model. (D) The distance between AR-C hydroxy group and D302 oxygen atoms (OD1 and OD2) in the simulation with the S-enantiomer (pink) and R-enantiomer (blue).
Figure 9. Position of AR-C155858 after simulation in the intermediate conformation(A) S-AR-C155858 was docked into the intermediate conformation of MCT1 and the best scoring solution taken for simulation in the WT model (left, 200 ns) or MCT1 model containing the mutations S364G and F360Y (right, 164 ns). (B) The position of S-AR-C155858 or R-AR-C155858 after 200 ns simulation in the WT MCT1 model. The same initial position and conformation of AR-C155858 was used as the start for all simulations.
Figure 10. Schematic diagram showing the proposed mechanism of inhibition by AR-C155858AR-C155858 crosses the plasma membrane to enter MCT1 in the inward-open conformation. An intermediate conformation is adopted, allowing binding of AR-C155858 by interaction with residues in the intracellular half including Asn147 (helix 5), Arg306 (helix 8) and Ser364 (helix 10). A further conformational change then allows movement of AR-C155858 further into the channel of MCT1, interacting with residues in the extracellular half including Lys38 (helix 1), Asp302 (helix 8), Phe360 (helix 10), Lys274 and Ser278 (helix 7). Note that the R- and S-enantiomers behave identically in the initial binding (Figure 9B) but only the S-enantiomer binds tightly in the final conformation (Figure 8).
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