Na+鈥揝ubstrate Coupling in the Multidrug Antiporter NorM Probed with a Spin-Labeled Substrate
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- 作者：P. Ryan Steed ; Richard A. Stein ; Smriti Mishra ; Michael C. Goodman ; Hassane S. Mchaourab
- 刊名：Biochemistry
- 出版年：2013
- 出版时间：August 27, 2013
- 年：2013
- 卷：52
- 期：34
- 页码：5790-5799
- 全文大小：424K
- 年卷期：v.52,no.34(August 27, 2013)
- ISSN：1520-4995
文摘
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鈥揺lectron 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+鈥揹rug coupling during antiport.