In vivo FRET analyses reveal a role of ATP hydrolysis-associated conformational changes in human P-glycoprotein.
J Biol Chem
; 295(15): 5002-5011, 2020 04 10.
Article
en En
| MEDLINE
| ID: mdl-32111736
ABSTRACT
P-glycoprotein (P-gp; also known as MDR1 or ABCB1) is an ATP-driven multidrug transporter that extrudes various hydrophobic toxic compounds to the extracellular space. P-gp consists of two transmembrane domains (TMDs) that form the substrate translocation pathway and two nucleotide-binding domains (NBDs) that bind and hydrolyze ATP. At least two P-gp states are required for transport. In the inward-facing (pre-drug transport) conformation, the two NBDs are separated, and the two TMDs are open to the intracellular side; in the outward-facing (post-drug transport) conformation, the NBDs are dimerized, and the TMDs are slightly open to the extracellular side. ATP binding and hydrolysis cause conformational changes between the inward-facing and the outward-facing conformations, and these changes help translocate substrates across the membrane. However, how ATP hydrolysis is coupled to these conformational changes remains unclear. In this study, we used a new FRET sensor that detects conformational changes in P-gp to investigate the role of ATP binding and hydrolysis during the conformational changes of human P-gp in living HEK293 cells. We show that ATP binding causes the conformational change to the outward-facing state and that ATP hydrolysis and subsequent release of γ-phosphate from both NBDs allow the outward-facing state to return to the original inward-facing state. The findings of our study underscore the utility of using FRET analysis in living cells to elucidate the function of membrane proteins such as multidrug transporters.
Palabras clave
Texto completo:
1
Banco de datos:
MEDLINE
Asunto principal:
Conformación Proteica
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Adenosina Trifosfato
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Miembro 1 de la Subfamilia B de Casetes de Unión a ATP
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Transferencia Resonante de Energía de Fluorescencia
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Multimerización de Proteína
Tipo de estudio:
Risk_factors_studies
Límite:
Humans
Idioma:
En
Revista:
J Biol Chem
Año:
2020
Tipo del documento:
Article
País de afiliación:
Japón