Specificity determinants in small multidrug transporters

Multiple-antibiotic resistance has become a major global public health concern, and to overcome this problem, it is necessary to understand the resistance mechanisms that allow survival of the microorganisms at the molecular level. One mechanism responsible for such resistance involves active removal of the antibiotic from the pathogen cell by MDTs (multi drug transporters). A prominent MDT feature is their high polyspecificity allowing for a single transporter to confer resistance against a range of drugs. Here we present the molecular mechanism underlying substrate recognition in EmrE, a small MDT from Escherichia coli. EmrE is known to have a substrate preference for aromatic, cationic compounds, such as methyl viologen (MV²⁺). In this work, we use a combined bioinformatic and biochemical approach to identify one of the major molecular determinants involved in MV²⁺ transport and resistance. Replacement of an Ala residue with Ser in weakly resistant SMRs from Bacillus pertussis and Mycobacterium tuberculosis enables them to provide robust resistance to MV²⁺ and to transport MV²⁺ and has negligible effects on the interaction with other substrates. This shows that the residue identified herein is uniquely positioned in the binding site so as to be exclusively involved in the mediating of MV²⁺ transport and resistance, both in EmrE and in other homologues. This work provides clues toward uncovering how specificity is achieved within the binding pocket of a polyspecific transporter that may open new possibilities as to how these transporters can be manipulated to bind a designed set of drugs.