Abstract
MdfA from Escherichia coli is a prototypical secondary multi-drug (Mdr) transporter that exchanges drugs for protons. MdfA-mediated drug efflux is driven by the proton gradient and enabled by conformational changes that accompany the recruitment of drugs and their release. In this work, we applied distance measurements by W-band double electron-electron resonance (DEER) spectroscopy to explore the binding of mito-TEMPO, a nitroxide-labeled substrate analog, to Gd(III)-labeled MdfA. The choice of Gd(III)-nitroxide DEER enabled measurements in the presence of excess of mito-TEMPO, which has a relatively low affinity to MdfA. Distance measurements between mito-TEMPO and MdfA labeled at the periplasmic edges of either of three selected transmembrane helices (TM3(101), TM5(168), and TM9(310)) revealed rather similar distance distributions in detergent micelles (n-dodecyl-β-d-maltopyranoside, DDM)) and in lipid nanodiscs (ND). By grafting the predicted positions of the Gd(III) tag on the inward-facing (I(f)) crystal structure, we looked for binding positions that reproduced the maxima of the distance distributions. The results show that the location of the mito-TEMPO nitroxide in DDM-solubilized or ND-reconstituted MdfA is similar (only 0.4 nm apart). In both cases, we located the nitroxide moiety near the ligand binding pocket in the I(f) structure. However, according to the DEER-derived position, the substrate clashes with TM11, suggesting that for mito-TEMPO-bound MdfA, TM11 should move relative to the I(f) structure. Additional DEER studies with MdfA labeled with Gd(III) at two sites revealed that TM9 also dislocates upon substrate binding. Together with our previous reports, this study demonstrates the utility of Gd(III)-Gd(III) and Gd(III)-nitroxide DEER measurements for studying the conformational behavior of transporters.