The N-terminal domain of an archaeal multidrug and toxin extrusion (MATE) transporter mediates proton coupling required for prokaryotic drug resistance.
J Biol Chem
; 294(34): 12807-12814, 2019 08 23.
Article
en En
| MEDLINE
| ID: mdl-31289123
ABSTRACT
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.
Palabras clave
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Protones
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Rodaminas
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Proteínas Arqueales
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Pyrococcus furiosus
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Farmacorresistencia Bacteriana
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Escherichia coli
/
Antineoplásicos
Tipo de estudio:
Prognostic_studies
Idioma:
En
Revista:
J Biol Chem
Año:
2019
Tipo del documento:
Article