Structures of a Na+-coupled, substrate-bound MATE multidrug transporter

Abstract

Multidrug transporters belonging to the multidrug and toxic compound extrusion (MATE) family expel dissimilar lipophilic and cationic drugs across cell membranes by dissipating a preexisting Na(+) or H(+) gradient. Despite its clinical relevance, the transport mechanism of MATE proteins remains poorly understood, largely owing to a lack of structural information on the substrate-bound transporter. Here we report crystal structures of a Na(+)-coupled MATE transporter NorM from Neisseria gonorrheae in complexes with three distinct translocation substrates (ethidium, rhodamine 6G, and tetraphenylphosphonium), as well as Cs(+) (a Na(+) congener), all captured in extracellular-facing and drug-bound states. The structures revealed a multidrug-binding cavity festooned with four negatively charged amino acids and surprisingly limited hydrophobic moieties, in stark contrast to the general belief that aromatic amino acids play a prominent role in multidrug recognition. Furthermore, we discovered an uncommon cation-π interaction in the Na(+)-binding site located outside the drug-binding cavity and validated the biological relevance of both the substrate- and cation-binding sites by conducting drug resistance and transport assays. Additionally, we uncovered potential rearrangement of at least two transmembrane helices upon Na(+)-induced drug export. Based on our structural and functional analyses, we suggest that Na(+) triggers multidrug extrusion by inducing protein conformational changes rather than by directly competing for the substrate-binding amino acids. This scenario is distinct from the canonical antiport mechanism, in which both substrate and counterion compete for a shared binding site in the transporter. Collectively, our findings provide an important step toward a detailed and mechanistic understanding of multidrug transport.

Fig. 1.

Structure of NorM-NG-monobody complex. (A and B) Structure of NorM-NG monobody complex as viewed from the membrane plane. The views in A and B are related by ∼180° rotation around the membrane normal. The amino (residues 5–230) and carboxyl (residues 231–459) halves of NorM-NG are colored cyan and yellow, respectively. Monobody is shown as a magenta ribbon and bound TPP as magenta sticks. (C) The arrangement of transmembrane helices in NorM-NG as viewed from the periplasmic side. (D) NorM-NG surface as viewed from the periplasmic side, which is colored according to electrostatic potentials from −20 (red) to +20 kTe⁻¹ (blue).

Fig. 2.

Structure of the multidrug-binding site. (A) NorM-NG is drawn as a ribbon, whereas the substrates are in stick representation. (B–D) Closeup of the binding site for TPP, ethidium (ET), and R6G, respectively. Amino acids within 4.5 Å (given the ∼0.6-Å coordinate errors at current resolutions) of the substrate are illustrated as sticks. L3-4 was omitted in B and C for clarity.

Fig. 3.

Na⁺-induced protein conformational changes. (A) Structural overlay of NorM-NG (cyan and yellow, PDB ID code 4HUK) and NorM-VC (gray, PDB ID code 3MKU) (9). TPP (stick model) is colored magenta; red arrows highlight the rearrangement of TM7 and TM8 relative to TM10. (B) Cation-bound structure of NorM-NG (purplish blue ribbon, PDB ID code 4HUL). Cs⁺ (green sphere) is overlaid with a difference isomorphous Fourier map (magenta mesh) contoured at 6σ. Red arrows indicate proposed movement of TM7 and TM8 toward TM10. TPP (magenta) taken from the TPP-bound structure (PDB 4HUK) is shown in stick representation to indicate the substrate-binding site. (C) Hypothetical Na⁺ (gray sphere) coordination arrangement that corresponds to state 3 in Fig. 4. Relevant amino acids are depicted as stick models and NorM-NG is colored gray.

Fig. 4.

Proposed antiport mechanism. Na⁺ (green circle) binds to a cation-free, drug-bound transporter (state 1) and elicits the movement of TM7 and TM8 (red arrow) in the cation-bound, drug-bound protein (state 2), causing the drug to dissociate. The cation-bound, drug-free transporter (state 3) then switches to the inward-facing conformation (state 4), before it binds another drug molecule (magenta). Drug-binding triggers the movement of TM7 and TM8 (red arrow), thereby weakening the Na⁺ binding (state 5). Na⁺ releases into the cytoplasm in an inward-facing, drug-bound transporter (state 6), and the transporter returns to the outward-facing, drug-bound conformation (state 1) to complete the transport cycle. Our cation-free, drug-bound NorM-NG structures (PDB ID codes 4HUK, 4HUM, and 4HUN) represent state 1, whereas the cation-bound NorM-NG (PDB ID code 4HUL) and NorM-VC structures (PDB ID codes 3MKU and 3MKT) emulate state 2 and state 3, respectively. TM1 and TM2 are simplified as a cyan stick, TM7 and TM8 as a thick yellow stick, and TM10 as a thin yellow stick.