Journal
BIOCHEMISTRY
Volume 52, Issue 34, Pages 5790-5799Publisher
AMER CHEMICAL SOC
DOI: 10.1021/bi4008935
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Funding
- National Institutes of Health [F32 GM100569, R01 GM077659]
- NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES [F32GM100569, R01GM077659] Funding Source: NIH RePORTER
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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 rnicromolar 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.
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