Structure of a cation-bound multidrug and toxic compound extrusion transporter

Abstract

Transporter proteins from the MATE (multidrug and toxic compound extrusion) family are vital in metabolite transport in plants, directly affecting crop yields worldwide. MATE transporters also mediate multiple-drug resistance (MDR) in bacteria and mammals, modulating the efficacy of many pharmaceutical drugs used in the treatment of a variety of diseases. MATE transporters couple substrate transport to electrochemical gradients and are the only remaining class of MDR transporters whose structure has not been determined. Here we report the X-ray structure of the MATE transporter NorM from Vibrio cholerae determined to 3.65 Å, revealing an outward-facing conformation with two portals open to the outer leaflet of the membrane and a unique topology of the predicted 12 transmembrane helices distinct from any other known MDR transporter. We also report a cation-binding site in close proximity to residues previously deemed critical for transport. This conformation probably represents a stage of the transport cycle with high affinity for monovalent cations and low affinity for substrates.

Figure 1. NorM-VC model

a, Front and b, back views of NorM-VC, with extracellular and cytoplasmic sides indicated. Stereo views presented in Fig. S1b. c, Extracellular view, with TM helices 1-12 marked. The internal cavity opens to the extracellular space and is occluded on the cytoplasmic side. The molecule is colored using a rainbow gradient from the N- (blue) to C-terminus (red).

Figure 2. Validation of the NorM-VC model, showing the positions of the 16 mercury-labeled cysteine mutants

a, Front and b, back views of a NorM-VC monomer (gray backbone) with isomorphous difference Fourier peaks, and mutated residues colored. Stereo views presented in Fig. S7. c, Topological comparison between MATE (left) and MFS (right) transporters from the cytoplasmic side. The C-terminal helix (residues 450-461) is removed for clarity and position of cation (Na⁺) is indicated. In MATE transporters; TM1 is between TMs 5 and 8, and TM7 is between TMs 3 and 11. The portals facing the membrane are formed between TMs 1 and 8, and TMs 2 and 7. In contrast, the MFS has TM1 between TMs 4, 5 and 6. Likewise, TM7 is between TMs 10, 11 and 12. d, x-ray structures of NorM, and the MFS transporter EmrD.

Figure 3. Electrostatic potential surface representation of NorM-VC

a, Views of the portals formed by TMs 1 and 8 and b, TMs 2 and 7, opening to the outer membrane leaflet highlighted and marked by dashed lines (yellow). The same views are shown in Fig. 1a, b. The surface of NorM is colored by amino acid residue charge ranging from blue (positive) to red (negative). Hydrophobic and aromatic residues are colored in white. TM regions embedded within the lipid bilayer are indicated. Stereo views are presented in Fig. S9.

Figure 4. The cation-binding site of NorM-VC and mechanism of transport

a, Cation-binding site superimposed with isomorphous difference Fourier map calculated between Crystal 2 (containing Rb⁺) and Crystal 3 (native) (Table S3), revealing a Rb⁺ peak at 5.5 σ (red mesh). b, A 5.0 σ peak (orange mesh) revealing binding of Cs⁺ in the same view as a, calculated between Crystal 4 (containing Cs⁺) and Crystal 3 (native). Stereo views presented in Fig. S12. c, Proposed transport mechanism: In the outward-facing conformation, cation (yellow) binds at a conserved site (blue oval; step 1). Cation binding induces structural changes to the inward-facing conformation (step 2), which is competent to bind substrate (organic cation in green) from the inner membrane leaflet or cytoplasm. Substrate binding causes structural changes back to the outward-facing conformation (step 3), allowing export and cation binding.