Conserved binding site in the N-lobe of prokaryotic MATE transporters suggests a role for Na+ in ion-coupled drug efflux

Emel Ficici,Wenchang Zhou,Derek P Claxton,Osamu Nureki,Hassane S Mchaourab,José D Faraldo-Gómez,Steven Castellano,Robyn Stix,Tsukasa Kusakizako

doi:10.1016/j.jbc.2021.100262

Abstract

In both prokaryotes and eukaryotes, multidrug and toxic-compound extrusion (MATE) transporters catalyze the efflux of a broad range of cytotoxic compounds, including human-made antibiotics and anticancer drugs. MATEs are secondary-active antiporters, i.e., their drug-efflux activity is coupled to, and powered by, the uptake of ions down a preexisting transmembrane electrochemical gradient. Key aspects of this mechanism, however, remain to be delineated, such as its ion specificity and stoichiometry. We previously revealed the existence of a Na+-binding site in a MATE transporter from Pyroccocus furiosus (PfMATE) and hypothesized that this site might be broadly conserved among prokaryotic MATEs. Here, we evaluate this hypothesis by analyzing VcmN and ClbM, which along with PfMATE are the only three prokaryotic MATEs whose molecular structures have been determined at atomic resolution, i.e. better than 3 Å. Reinterpretation of existing crystallographic data and molecular dynamics simulations indeed reveal an occupied Na+-binding site in the N-terminal lobe of both structures, analogous to that identified in PfMATE. We likewise find this site to be strongly selective against K+, suggesting it is mechanistically significant. Consistent with these computational results, DEER spectroscopy measurements for multiple doubly-spin-labeled VcmN constructs demonstrate Na+-dependent changes in protein conformation. The existence of this binding site in three MATE orthologs implicates Na+ in the ion-coupled drug-efflux mechanisms of this class of transporters. These results also imply that observations of H+-dependent activity likely stem either from a site elsewhere in the structure, or from H+ displacing Na+ under certain laboratory conditions, as has been noted for other Na+-driven transport systems.

Highlights

The development of multidrug resistance (MDR) in major human microbial pathogens is an increasingly alarming publichealth threat
-called MDR efflux pumps are the class of membrane transporters that provide this kind of defense
In two recent studies of PfMATE and NorM-VC, we used molecular dynamics (MD) simulations [33] and double electron–electron resonance (DEER) spectroscopy [34] to identify a Na+ binding site in the N-lobe of these transporters; we showed that it reflects a highly conserved sequence motif among prokaryotic multidrug and toxiccompound extrusion (MATE) [33]

Summary

Introduction

The development of multidrug resistance (MDR) in major human microbial pathogens is an increasingly alarming publichealth threat. -called MDR efflux pumps are the class of membrane transporters that provide this kind of defense These transporters deplete the cytosolic concentration of human-made antimicrobial drugs as well as natural immunologic compounds, protecting their intracellular targets. Ion coupling in MATE multi-drug transporters of seemingly outward-open MATEs have been resolved at varying resolutions [14,15,16,17,18,19,20,21,22] In these structures the N- and Clobes adopt a V-like conformation, i.e., they are in contact only on the intracellular side and project away from each other on the extracellular side, exposing a large cavity to the exterior. MATEs are driven either by Na+ or H+

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Conclusion