Protonation drives the conformational switch in the multidrug transporter LmrP

Abstract

Multidrug antiporters of the Major Facilitator Superfamily couple proton translocation to the extrusion of cytotoxic molecules. The conformational changes that underlie the transport cycle and the structural basis of coupling of these transporters have not been elucidated. Here we utilized extensive Double Electron Electron Resonance measurements to uncover the conformational equilibrium of LmrP, a multidrug transporter from L. lactis, and to investigate how protons and ligands shift this equilibrium to enable transport. We find that the transporter switches between outward-open and outward-closed conformations depending on the protonation states of specific acidic residues forming a transmembrane protonation relay. Our data can be framed in a model of transport wherein substrate binding initiates the transport cycle by opening the extracellular side. Subsequent protonation of membrane-embedded acidic residues induces substrate release to the extracellular side and triggers a cascade of conformational changes that concludes in proton release to the intracellular side.

Conformational cycle and ion-coupling mechanism of the Na + /hydantoin transporter Mhp1

“…This methodology has been successfully applied to define coupled conformational cycles for a number of transporter classes (13,(26)(27)(28)(29)(30)(31)(32). We find that patterns of distance distributions between pairs of spin labels monitoring the intra-and extracellular sides of Mhp1 are consistent with isomerization between the crystallographic inward-and outward-facing conformations.…”

Emulating proton-induced conformational changes in the vesicular monoamine transporter VMAT2 by mutagenesis

“…Protonation has been shown to induce conformational changes in other protoncoupled MFS transporters. For example, in FucP, protonation of a key carboxyl was suggested to facilitate the transition from extracellular-to cytoplasm-facing (45,46); in EmrD, decreasing the pH led to opening of the cytoplasmic cleft (47); in LacY, deprotonation enabled a conformational change of the empty transporter (10,48,49); and a recent study suggested a protonation event as a molecular switch between conformations in LmrP (50). Given that mutating D33 to Asn resulted in a very similar behavior to that of the gating mutants (Fig.…”

Functionally Important Carboxyls in a Bacterial Homologue of the Vesicular Monoamine Transporter (VMAT)

“…In two other well characterized but more distant MFS multidrug antiporters from Gram-positive bacteria, LmrP and QacA, membrane-embedded carboxylic acids in various TMs have been shown to play a role in substrate binding and proton translocation, although the location of the carboxylic amino acids is not conserved (62)(63)(64)(65)(66). Among the well characterized MFS antiporters, the highest similarity and, presumably, mechanism is indeed observed between BbMAT and rVMAT2 (Fig.…”