title: Identification of the High-affinity Substrate-binding Site of the Multidrug and Toxic Compound Extrusion (MATE) Family Transporter from Pseudomonas stutzeri.
abstract: Multidrug and toxic compound extrusion (MATE) transporters exist in all three domains of life. They confer multidrug resistance by utilizing H(+) or Na(+) electrochemical gradients to extrude various drugs across the cell membranes. The substrate binding and the transport mechanism of MATE transporters is a fundamental process but so far not fully understood. Here we report a detailed substrate binding study of NorM_PS, a representative MATE transporter from Pseudomonas stutzeri Our results indicate that NorM_PS is a proton-dependent multidrug efflux transporter. Detailed binding studies between NorM_PS and 4',6-diamidino-2-phenylindole (DAPI) were performed by isothermal titration calorimetry (ITC), differential scanning calorimetry (DSC), and spectrofluorometry. Two exothermic binding events were observed from ITC data, and the high-affinity event was directly correlated with the extrusion of DAPI. The affinities are about 1 μm and 0.1 mm for the high and low affinity binding, respectively. Based on our homology model of NorM_PS, variants with mutations of amino acids that are potentially involved in substrate binding, were constructed. By carrying out the functional characterization of these variants, the critical amino acid residues (Glu-257 and Asp-373) for high-affinity DAPI binding were determined. Taken together, our results suggest a new substrate-binding site for MATE transporters.
authors.affiliation: From the Tianjin University, School of Chemical Engineering and Technology, State Key Laboratory for Chemical Engineering, Collaborative Innovation Center of Chemical Science and Chemical Engineering, 300072 Tianjin, China and the Department of Molecular Membrane Biology, Max Planck Institute of Biophysics, Max-von-Laue-Strasse 3, 60438 Frankfurt am Main, Germany.
authors.forename: Laiyin
authors.initials: L
authors.lastname: Nie
authors.affiliation: the Department of Molecular Membrane Biology, Max Planck Institute of Biophysics, Max-von-Laue-Strasse 3, 60438 Frankfurt am Main, Germany.
authors.forename: Ernst
authors.initials: E
authors.lastname: Grell
authors.affiliation: the Department of Molecular Membrane Biology, Max Planck Institute of Biophysics, Max-von-Laue-Strasse 3, 60438 Frankfurt am Main, Germany.
authors.forename: Viveka Nand
authors.initials: VN
authors.lastname: Malviya
authors.affiliation: the Department of Molecular Membrane Biology, Max Planck Institute of Biophysics, Max-von-Laue-Strasse 3, 60438 Frankfurt am Main, Germany.
authors.forename: Hao
authors.initials: H
authors.lastname: Xie
authors.affiliation: From the Tianjin University, School of Chemical Engineering and Technology, State Key Laboratory for Chemical Engineering, Collaborative Innovation Center of Chemical Science and Chemical Engineering, 300072 Tianjin, China and wangjkch@tju.edu.cn.
authors.forename: Jingkang
authors.initials: J
authors.lastname: Wang
authors.affiliation: the Department of Molecular Membrane Biology, Max Planck Institute of Biophysics, Max-von-Laue-Strasse 3, 60438 Frankfurt am Main, Germany hartmut.michel@biophys.mpg.de.
authors.forename: Hartmut
authors.initials: H
authors.lastname: Michel
mesh_terms: D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D024901:Drug Resistance, Multiple, Bacterial; D007211:Indoles / Q000737:chemistry* / Q000378:metabolism; D020125:Mutation, Missense; D044223:Pseudomonas stutzeri / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D002352:Carrier Proteins; D007211:Indoles; C007293:DAPI
keywords: DAPI-binding site; binding affinity; isothermal titration calorimetry (ITC); ligand-binding protein; membrane biophysics; membrane protein; multidrug transporter