Inward-facing conformation of a multidrug resistance MATE family transporter

Abstract

Multidrug and toxic compound extrusion (MATE) transporters mediate excretion of xenobiotics and toxic metabolites, thereby conferring multidrug resistance in bacterial pathogens and cancer cells. Structural information on the alternate conformational states and knowledge of the detailed mechanism of MATE transport are of great importance for drug development. However, the structures of MATE transporters are only known in V-shaped outward-facing conformations. Here, we present the crystal structure of a MATE transporter from Pyrococcus furiosus (PfMATE) in the long-sought-after inward-facing state, which was obtained after crystallization in the presence of native lipids. Transition from the outward-facing state to the inward-facing state involves rigid body movements of transmembrane helices (TMs) 2-6 and 8-12 to form an inverted V, facilitated by a loose binding of TM1 and TM7 to their respective bundles and their conformational flexibility. The inward-facing structure of PfMATE in combination with the outward-facing one supports an alternating access mechanism for the MATE family transporters.

Keywords: MATE transporter; inward-facing conformation; lipids; membrane protein structure; multidrug resistance.

Fig. 1.

Crystal structures of PfMATE in two distinct conformations. (A) Side view of PfMATE representing outward-facing (OFC) and inward-facing (IFC) conformations. Ribbon models of the N-lobe (TM1–TM6) and the C-lobe (TM7–TM12) are shown in pink and blue, respectively. The dashed lines represent the borders of the lipid bilayer. (B, Left) Structural superposition of the N-lobes and C-lobes of two states, indicating a substantial alteration of TM1 in the N-lobe and TM7 in the C-lobe, whereas the remaining helices undergo a rigid body movement. (B, Right) Structural alignment of the N-lobe and C-lobe of the IFC shows their symmetrical arrangement. (C) Surface representation of the OFC and IFC showing the central binding cavity from the top and in cross-section.

Fig. 2.

Sodium ion-binding site in PfMATE and water accessibility. (A) Water occupancy of the binding-site cavity (blue surface) in simulations of inward-facing and outward-facing conformations. Water access to the cavity is restricted to the inside and outside, respectively. (B) Distance between the sodium ion-binding site (carboxyl oxygen atoms of Asp41) and a Na⁺ that binds spontaneously during MD simulations of the outward-facing conformation (OFC) state (Top) and that remained bound in the inward-facing conformation (IFC) state (Bottom). Sodium ion-binding site in the IFC state, based on the crystal structure (PDB ID code 6FHZ) (C) and the representative MD snapshot (D). The residues labeled and shown by a stick model coordinate the bound Na⁺, depicted as an orange sphere. The purple mesh represents the Fo-Fc electron density peak, which is tentatively assigned to the Na⁺ ion. The sodium ion-binding site in the OFC state, based on the crystal structure (PDB ID code 6HFB) (E) and an MD snapshot of the fully coordinated sodium ion (taken from the simulation at 700 ns) (F). The coordinating residues are shown in stick representation, whereas the blue mesh shows the anomalous peak for Cs⁺.

Fig. 3.

Spontaneous binding and reorientation of an archaeal lipid in MD simulations of PfMATE. (Left) Side view of the representative MD snapshot showing the interaction of PfMATE (ribbons; N-lobe in pink and C-lobe in blue) with the archaeal-type DPG lipid (stick model) that entered the cavity from the outside leaflet at the top and spontaneously flipped its orientation. (Right) Zoom-in view highlights residues contacting the lipid. MD snapshots have been taken from the end of the 1.5-μs simulation.