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  Identification of the High-affinity Substrate-binding Site of the Multidrug and Toxic Compound Extrusion (MATE) Family Transporter from Pseudomonas stutzeri

Laiyin, N., Grell, E., Malviya, V. N., Xie, H., Wang, J., & Michel, H. (2016). Identification of the High-affinity Substrate-binding Site of the Multidrug and Toxic Compound Extrusion (MATE) Family Transporter from Pseudomonas stutzeri. The Journal of Biological Chemistry, 291(30), 15503-15514. doi:10.1074/jbc.M116.728618.

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Laiyin, Nie1, 2, Author           
Grell, Ernst1, Author           
Malviya, Viveka Nand1, Author           
Xie, Hao1, Author           
Wang, Jiangkang2, Author
Michel, Hartmut1, Author                 
Affiliations:
1Department of Molecular Membrane Biology, Max Planck Institute of Biophysics, Max Planck Society, ou_2068290              
2Tianjin 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, ou_persistent22              

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 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.

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Language(s): eng - English
 Dates: 2016-03-222016-05-272016-05-272016-07-22
 Publication Status: Published in print
 Pages: 12
 Publishing info: -
 Table of Contents: -
 Rev. Type: Peer
 Identifiers: DOI: 10.1074/jbc.M116.728618
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Title: The Journal of Biological Chemistry
  Other : JBC
Source Genre: Journal
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Publ. Info: Baltimore, etc. : American Society for Biochemistry and Molecular Biology [etc.]
Pages: - Volume / Issue: 291 (30) Sequence Number: - Start / End Page: 15503 - 15514 Identifier: ISSN: 0021-9258
CoNE: https://pure.mpg.de/cone/journals/resource/954925410826_1