Multidrug Transporter LmrP allows Relocation of Catalytic Carboxylates [Membrane Biology]

December 2nd, 2015 by Tong, Z., Ding, N., Neuberger, A., van Veen, H. W.

One of the important mechanisms of drug resistance in cells is based on active drug extrusion by multidrug efflux transporters. LmrP is a secondary-active major facilitator superfamily multidrug transporter from the non-pathogenic, food-grade, Gram-positive bacterium Lactococcus lactis, which is similar to major facilitator superfamily multidrug transporters in pathogenic bacteria. LmrP mediates multidrug efflux in a proton motive force-dependent fashion by catalyzing electrogenic substrate/proton antiport. For this purpose, LmrP contains a number of carboxyl residues which, together with polar and aromatic residues, are organized in two clusters on the surface of a large interior chamber. To further investigate the functional role of these catalytic carboxylates and clusters in LmrP, we changed the composition of Cluster 1 by removal of Glu-327 and relocation of this carboxyl to 6 positions in the interior chamber. Among the tested positions, we found that the reinsertion at positions Thr-331 and Ala-355 in proximity of Cluster 2 restored wild-type energetics and kinetics of propidium and/or ethidium transport. Moreover, the introduction of T331E in wild-type LmrP enhanced the rate of active propidium efflux due to the ability of T331E to act as an additional proton-binding group. This behavior is very different from substrate-dedicated transporters in which the location of catalytic carboxylates in the proton coupling reaction is very precise and crucial for enzyme activity. Our data uncover an intrinsic flexibility in the location of the key residues in LmrP that are responsible for proton/drug exchange.