title: Na⁺-substrate coupling in the multidrug antiporter norm probed with a spin-labeled substrate.
abstract: NorM of the multidrug and toxic compound extrusion (MATE) family of transporters couples the efflux of a broad range of hydrophobic molecules to an inward Na⁺ gradient across the cell membrane. Several crystal structures of MATE transporters revealed distinct substrate binding sites leading to differing models of the mechanism of ion-coupled substrate extrusion. In the experiments reported here, we observed that a spin-labeled derivative of daunorubicin, Ruboxyl, is transported by NorM from Vibrio cholerae. It is therefore ideal for characterizing mechanistically relevant binding interactions with NorM and directly addressing the coupling of ion and drug binding. Fluorescence and electron paramagnetic resonance experiments revealed that Ruboxyl binds to NorM with micromolar affinity and becomes immobilized upon binding, even in the presence of Na⁺. Using double electron-electron resonance spectroscopy, we determined that Ruboxyl binds to a single site on the periplasmic side of the protein. The presence of Na⁺ did not translocate the substrate to a second site as previously proposed. These experiments surprisingly show that Na⁺ does not affect the affinity or location of the substrate binding site on detergent-solubilized NorM, thus suggesting that additional factors beyond simple mutual exclusivity of binding, such as the presence of a Na⁺ gradient across the native membrane, govern Na⁺-drug coupling during antiport.
authors.affiliation: Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, Tennessee 37232, United States.
authors.forename: P Ryan
authors.initials: PR
authors.lastname: Steed
authors.forename: Richard A
authors.initials: RA
authors.lastname: Stein
authors.forename: Smriti
authors.initials: S
authors.lastname: Mishra
authors.forename: Michael C
authors.initials: MC
authors.lastname: Goodman
authors.forename: Hassane S
authors.initials: HS
authors.lastname: McHaourab
mesh_terms: D017920:Antiporters / Q000378:metabolism*; D001426:Bacterial Proteins / Q000378:metabolism*; D001665:Binding Sites / Q000187:drug effects; D003630:Daunorubicin / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology; D004926:Escherichia coli / Q000187:drug effects; D011485:Protein Binding; D012964:Sodium / Q000378:metabolism* / Q000494:pharmacology; D013113:Spin Labels; D014734:Vibrio cholerae / Q000737:chemistry
chemical_list: D017920:Antiporters; D001426:Bacterial Proteins; C114619:NorM protein, bacteria; D013113:Spin Labels; C042480:ruboxyl; D012964:Sodium; D003630:Daunorubicin
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