The N-terminal domain of an archaeal multidrug and toxin extrusion (MATE) transporter mediates proton coupling required for prokaryotic drug resistance
| Autor: | Hassane S. Mchaourab, Derek P. Claxton, Kevin L. Jagessar |
|---|---|
| Rok vydání: | 2019 |
| Předmět: |
0301 basic medicine
Archaeal Proteins Allosteric regulation Antineoplastic Agents medicine.disease_cause Biochemistry 03 medical and health sciences Ion binding Membrane Biology Drug Resistance Bacterial Escherichia coli medicine Molecular Biology Cell Proliferation chemistry.chemical_classification 030102 biochemistry & molecular biology biology Rhodamines Transporter Cell Biology biology.organism_classification Amino acid Pyrococcus furiosus Multiple drug resistance 030104 developmental biology chemistry Biophysics Efflux Protons |
| Zdroj: | J Biol Chem |
| ISSN: | 0021-9258 |
| DOI: | 10.1074/jbc.ra119.009195 |
| Popis: | As a contributor to multidrug resistance, the family of multidrug and toxin extrusion (MATE) transporters couples the efflux of chemically dissimilar compounds to electrochemical ion gradients. Although divergent transport mechanisms have been proposed for these transporters, previous structural and functional analyses of members of the MATE subfamily DinF suggest that the N-terminal domain (NTD) supports substrate and ion binding. In this report, we investigated the relationship of ligand binding within the NTD to the drug resistance mechanism of the H(+)-dependent MATE from the hyperthermophilic archaeon Pyrococcus furiosus (PfMATE). To facilitate this study, we developed a cell growth assay in Escherichia coli to characterize the resistance conferred by PfMATE to toxic concentrations of the antimicrobial compound rhodamine 6G. Expression of WT PfMATE promoted cell growth in the presence of drug, but amino acid substitutions of conserved NTD residues compromised drug resistance. Steady-state binding analysis with purified PfMATE indicated that substrate affinity was unperturbed in these NTD variants. However, exploiting Trp fluorescence as an intrinsic reporter of conformational changes, we found that these variants impaired formation of a unique H(+)-stabilized structural intermediate. These results imply that disruption of H(+) coupling is the origin of compromised toxin resistance in PfMATE variants. These findings support a model mechanism wherein the NTD mediates allosteric coupling to ion gradients through conformational changes to drive substrate transport in PfMATE. Furthermore, the results provide evidence for diverging transport mechanisms within a prokaryotic MATE subfamily. |
| Databáze: | OpenAIRE |
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