Structure of an engineered multidrug transporter MdfA reveals the molecular basis for substrate recognition

Abstract

MdfA is a prototypical H ⁺-coupled multidrug transporter that is characterized by extraordinarily broad substrate specificity. The involvement of specific H-bonds in MdfA-drug interactions and the simplicity of altering the substrate specificity of MdfA contradict the promiscuous nature of multidrug recognition, presenting a baffling conundrum. Here we show the X-ray structures of MdfA variant I239T/G354E in complexes with three electrically different ligands, determined at resolutions up to 2.2 Å. Our structures reveal that I239T/G354E interacts with these compounds differently from MdfA and that I239T/G354E possesses two discrete, non-overlapping substrate-binding sites. Our results shed new light on the molecular design of multidrug-binding and protonation sites and highlight the importance of often-neglected, long-range charge-charge interactions in multidrug recognition. Beyond helping to solve the ostensible conundrum of multidrug recognition, our findings suggest the mechanistic difference between substrate and inhibitor for any H ⁺-dependent multidrug transporter, which may open new vistas on curtailing efflux-mediated multidrug resistance.

Abstract

Hsin-Hui Wu et al. present the structure of the I239T/G354E variant of the MdfA multidrug transporter in complex with three different ligands. They find that this variant has two discrete, non-overlapping substrate binding sites, leading to differential interactions with ligands compared to wild-type MdfA.