Multidrug and toxin extrusion (MATE) family transporters are ubiquitous in life and excrete toxic compounds coupled to Na+/H+ influx. Although static structures of several MATE transporters are determined, the structural mechanism behind the ion-coupled transport has not been elucidated. To address this central issue in understanding the function of transporters, we conducted a structural analysis of Pyrococcus furiosus MATE (PfMATE) using solution-state NMR.
Our NMR analysis indicated that PfMATE adopts both inward-facing (IF) and outward-facing (OF) conformations in equilibrium, and predominantly exhibits the OF conformation in a deprotonated state (pH 6.8) but mostly adopts the IF conformation in a protonated state (pH 3.7).In contrast, substitutions of the intracellular protonation residues, Glu163 and, to a lesser extent, Asp165, shifted the equilibrium to the IF conformation, indicating the critical role of the intracellular protonation site(s) in regulation of the OF/IF conformational equilibrium. Importantly, We also found that the binding affinities of substrates, ethidium and norfloxacin, to the OF conformation are weak in the millimolar range, while those to the IF conformation are much stronger in mid-micromolar range.Furthermore, the substrate interaction with the IF conformation enhanced proton release from the intracellular protonation sites.
These results indicate that PfMATE is an affinity-directed substrate/H+-antiporter, in which substrate interactions primarily occur in the Glu163-protonated IF conformation and the subsequent proton release switches the conformation back to the OF conformation to ensure a proton-coupled compound excretion.