Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.08.31.275321v1?rss=1 Authors: Goldfarb, D., Bahrenberg, T., Yardeni, E. H., Feintuch, A., Bibi, E. Abstract: MdfA, a member of the major facilitator superfamily (MFS), is a multidrug/proton antiporter from E. coli that has been considered a model for secondary multidrug (Mdr) transporters. Its transport mechanism, driven by a proton gradient, is associated with conformational changes, which accompany the recruitment of drugs and their release. In this work, we applied double-electron electron resonance (DEER) spectroscopy to locate the binding site of one of its substrates, tetraphenylphosphonium (TPP) within available crystal structures. We carried out Gd(III)-nitroxide distance measurements between MdfA labeled with a Gd(III) tag and the TPP analog mito-TEMPO (bearing the nitroxide moiety). Data were obtained both for MdfA solubilized in detergent micelles (n-dodecyl-{beta}-D-maltopyranoside )DDM)), and reconstituted into lipid nanodiscs (ND). For both DDM and ND, the average position of the substrate at a neutral pH was found to be close to the ligand position in the If (inward facing) crystal structure, with the DDM environment exhibiting a somewhat better agreement than the ND environment. We therefore conclude that the If structure provides a good description for substrate-bound MdfA in DDM solution, while in ND the structure is slightly modified. A second binding site was found for the ND sample situated at the cytoplasmic side, towards the end of transmembrane helix 7 (TM7). In addition, we used DEER distance measurements on Gd(III) doubly labeled MdfA to track conformational changes within the periplasmic and cytoplasmic sides associated with substrate binding. We detected significant differences in the periplasmic side of MdfA, with the ND featuring a more closed conformation than in DDM, in agreement with earlier reports. The addition of TPP led to a noticeable conformational change in the periplasmic face in ND, attributed to a movement of TM10. This change was not observed in DDM. Copy rights belong to original authors. Visit the link for more info