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

Hsin-Hui Wu,Min Lu,Jindrich Symersky

doi:10.1038/s42003-019-0446-y

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.

Highlights

MdfA is a prototypical H+-coupled multidrug transporter that is characterized by extraordinarily broad substrate specificity
The best diffracting crystals of I239T/G354E were obtained in the presence of LDAO at pH 8
We found that the mutations of Y30, N33, D34, M58, L62, Y127, M146, L236, Q357, and F361 markedly reduced the ability of I239T/G354E to confer resistance against methyl viologen (MV), whereas the mutations of A150, S232, I239T, V335, L339, S350, and M353, most of which bind LDAO2, had little deleterious effect

Summary

Introduction

MdfA is a prototypical H+-coupled multidrug transporter that is characterized by extraordinarily broad substrate specificity. 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. A principal mechanism underpinning the unabated multidrug resistance is mediated by integral membrane proteins known as multidrug transporters[1,3]. MdfA exhibits an extremely broad spectrum of drug recognition and can couple the export of cationic, neutral, and zwitterionic compounds to the import of H+, with a drug/H+ stoichiometry of 1:1 MdfA can interact with two substrates simultaneously, one inhibits the transport of the other[20]

Methods

Results

Conclusion