The Conformational Dynamics of a Secondary Multidrug Transporter are Modulated by the Lipid Bilayer Composition

Abstract

LmrP, a Major Facilitator Superfamily (MFS) multidrug transporter from Lactococcus lactis, acts as a secondary antiporter, catalysing the extrusion of a spectrum of hydrophobic drugs by dissipating a proton gradient. It has evolved to bind and export structurally diverse cytotoxic substances from within the membrane, in contrast to the majority of MFS transporters specialized in the transport of a single soluble substrate, captured from the aqueous medium. Its transport mechanism could thus differ from the alternating access model of the lactose permease, the actual paradigm for all MFS transporters. Recently, we have shown that the protonation of conserved acidic residues is the driving force of the conformational transition, and that substrate binding initiates the cycle by catalysing proton passage (Masureel, Martens et al. 2014). While this analysis was performed on detergent solubilized protein, we now aim to investigate the role of the lipid bilayer in the conformational cycle. For this purpose, we performed Double Electron Electron Resonance (DEER) distance measurements on a library of labeled double-cysteine mutants monitoring both sides of the transporter. Spin labeled mutants were reconstituted in nanodiscs and the distance changes monitored upon changes in the pH or addition of substrate(s). We found that the pH-dependent equilibrium between the inside-open and outside-open conformations is strongly shifted in nanodiscs compared to detergent, suggesting that the pKa of the acidic residues is modified by the lipids. By combining specific lipidic compositions and mutagenesis, we are investigating whether such change in the dynamics are the results of direct phospholipid-protein interaction. Specific structural effects of given lipids are observed: small amounts of cardiolipin allow the conformational transition to occur at physiological pH. These findings could shed light on the mechanism of MFS multidrug transporters in general.