Biophys J. 2021 Mar 23:S0006-3495(21)00243-5. doi: 10.1016/j.bpj.2021.03.014. Online ahead of print.
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, which 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 (TM3101 ,TM5168 and TM9310) 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 (If) crystal structure, we looked for binding positions which 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 If 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 If structure. Additional DEER studies with MdfA labeled by 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.