RT Journal Article SR Electronic T1 Sodium and proton coupling in the conformational cycle of a MATE antiporter from Vibrio cholerae JF Proceedings of the National Academy of Sciences JO Proc Natl Acad Sci USA FD National Academy of Sciences SP E6182 OP E6190 DO 10.1073/pnas.1802417115 VO 115 IS 27 A1 Claxton, Derek P. A1 Jagessar, Kevin L. A1 Steed, P. Ryan A1 Stein, Richard A. A1 Mchaourab, Hassane S. YR 2018 UL http://www.pnas.org/content/115/27/E6182.abstract AB Transporters from the multidrug and toxic compound extrusion (MATE) superfamily protect the cell from cytotoxic molecules through an efflux mechanism that is dependent on ion electrochemical gradients. This study examined the role of specific residues in supporting conformational changes associated with ion and drug binding in NorM, an archetype of MATE transporters. The results show that a network of conserved residues in the N-terminal domain is critical for Na+- and H+-driven conformational changes, whereas residues in the C-terminal domain mediate drug binding. Informed by a correlation of conformational dynamics with transport activity, we propose a model that describes how conserved residues mediate ion-coupled structural changes underlying drug efflux.Secondary active transporters belonging to the multidrug and toxic compound extrusion (MATE) family harness the potential energy of electrochemical ion gradients to export a broad spectrum of cytotoxic compounds, thus contributing to multidrug resistance. The current mechanistic understanding of ion-coupled substrate transport has been informed by a limited set of MATE transporter crystal structures from multiple organisms that capture a 12-transmembrane helix topology adopting similar outward-facing conformations. Although these structures mapped conserved residues important for function, the mechanistic role of these residues in shaping the conformational cycle has not been investigated. Here, we use double-electron electron resonance (DEER) spectroscopy to explore ligand-dependent conformational changes of NorM from Vibrio cholerae (NorM-Vc), a MATE transporter proposed to be coupled to both Na+ and H+ gradients. Distance measurements between spin labels on the periplasmic side of NorM-Vc identified unique structural intermediates induced by binding of Na+, H+, or the substrate doxorubicin. The Na+- and H+-dependent intermediates were associated with distinct conformations of TM1. Site-directed mutagenesis of conserved residues revealed that Na+- and H+-driven conformational changes are facilitated by a network of polar residues in the N-terminal domain cavity, whereas conserved carboxylates buried in the C-terminal domain are critical for stabilizing the drug-bound state. Interpreted in conjunction with doxorubicin binding of mutant NorM-Vc and cell toxicity assays, these results establish the role of ion-coupled conformational dynamics in the functional cycle and implicate H+ in the doxorubicin release mechanism.