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??. 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 ... More
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??. 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.
