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

Laiyin Nie,Ernst Grell,Viveka Nand Malviya,Hao Xie,Jingkang Wang,Hartmut Michel

doi:10.1074/jbc.m116.728618

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.

Highlights

The ability of all living organisms to protect themselves against toxic substances is essential for their survival
The drug-hypersusceptible E. coli strain KAM32 overexpressing NorM_PS displays increased resistance to 6 toxic compounds, which demonstrates that the protein functions as a toxin extruder in vivo
NorM_PS exhibits a moderate level of cellular resistance against substrates (2– 4-fold), which is in accordance with former studies on multidrug and toxic compound extrusion (MATE) transporters [10, 25]

Summary

Results

Production and Purification of NorM_PS and Its Variants— Expression of NorM_PS could be detected in the cell lysates by Western blot analysis with the anti-polyhistidine antibody. Of all the tested variants (D38N, E257A, E257Q, E257D, and D373N), E257A showed an undetectable expression level under our standard conditions (Fig. 1a), and all other variants could be produced in KAM32 cells with similar amounts as the wild-type (WT) protein. A single peak in the size exclusion chromatography indicated that NorM_PS could be purified to homogeneity (Fig. 1c); the corresponding Coomassie-stained SDS-PAGE gel demonstrated a relatively high purity (Fig. 1b). The molecular mass of NorM_PS in detergent solution was determined to be 54.7 kDa, which corresponds well to the theoretical monomeric molar mass of 53.0 kDa. Surprisingly, the amount of detergent associated with NorM_PS was quite high, the mass ratio between ␤-DDM and NorM_PS (␦) was 2.82. Elevated MIC values were observed for DAPI, doxorubicin, daunorubicin, tetracycline, kanamycin, and gentamycin, implying that Escherichia coli KAM32 cells harboring NorM_PS exhibited a higher resistance to those toxic compounds.

Toxic Compounds

Physicochemical and Biochemical Aspects

Structural Aspects

Experimental Procedures