Structural basis for the blockade of MATE multidrug efflux pumps.

Martha Radchenko,Jindrich Symersky,Rongxin Nie,Min Lu

doi:10.1038/ncomms8995

Martha Radchenko, Jindrich Symersky + Show 2 more

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https://doi.org/10.1038/ncomms8995

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Abstract

Multidrug and toxic compound extrusion (MATE) transporters underpin multidrug resistance by using the H+ or Na+ electrochemical gradient to extrude different drugs across cell membranes. MATE transporters can be further parsed into the DinF, NorM and eukaryotic subfamilies based on their amino-acid sequence similarity. Here we report the 3.0 Å resolution X-ray structures of a protonation-mimetic mutant of an H+-coupled DinF transporter, as well as of an H+-coupled DinF and a Na+-coupled NorM transporters in complexes with verapamil, a small-molecule pharmaceutical that inhibits MATE-mediated multidrug extrusion. Combining structure-inspired mutational and functional studies, we confirm the biological relevance of our crystal structures, reveal the mechanistic differences among MATE transporters, and suggest how verapamil inhibits MATE-mediated multidrug efflux. Our findings offer insights into how MATE transporters extrude chemically and structurally dissimilar drugs and could inform the design of new strategies for tackling multidrug resistance.

Highlights

Multidrug and toxic compound extrusion (MATE) transporters underpin multidrug resistance by using the H þ or Na þ electrochemical gradient to extrude different drugs across cell membranes
We determined the structure of DinF-BHD40N to 3.5 Å resolution by using molecular replacement and multiple isomorphous replacement and anomalous scattering (MIRAS) phasing (Fig. 1, Supplementary Table 1)
Despite the substantial protein sequence similarity between DinF-BH and PfMATE (Supplementary Fig. 3), we found no structural proof for protonation-induced TM1-bending in the extracellular-facing DinF-BH

Summary

Introduction

Multidrug and toxic compound extrusion (MATE) transporters underpin multidrug resistance by using the H þ or Na þ electrochemical gradient to extrude different drugs across cell membranes. The X-ray structures of Na þ -dependent NorM transporters from Vibrio cholerae (NorM-VC)[6] and Neisseria gonorrhoeae (NorM-NG)[7], as well as H þ -dependent DinF transporters from Pyrococcus furiosus (PfMATE)[8] and Bacillus halodurans (DinF-BH)[9] have been reported Those crystal structures revealed a similar protein fold comprising 12 membrane-spanning segments (TM1-TM12), they suggested different arrangement of cation- and substrate-binding sites between the NorM and DinF proteins[8,9], hinting at substantial mechanistic diversity among the MATE transporters[9]. Combining crystallographic and biochemical studies, we shed new light on the mechanistic features that allow MATE transporters to extrude chemically and structurally different drugs, as well as the mechanism whereby verapamil inhibits mechanistically distinct DinF and NorM transporters

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