Conformational changes of P-glycoprotein by nucleotide binding

Guichun Wang, Roxana Pincheira, Mei Zhang, Jian-Ting Zhang

Research output: Contribution to journalArticle

56 Citations (Scopus)

Abstract

P-glycoprotein (Pgp) is a membrane protein that transports chemotherapeutic drugs, causing multidrug resistance in human cancer cells. Pgp is a member of the ATP-binding cassette superfamily and functions as a transport ATPase. It has been suggested that the conformation of Pgp changes in the catalytic cycle. In this study, we tested this hypothesis by using limited proteolysis as a tool to detect different conformational states trapped by binding of nucleotide ligands and inhibitors. Pgp has high basal ATPase activity; that is, ATP hydrolysis by Pgp is not rigidly associated with drug transport. This activity provides a convenient method for studying the conformational change of Pgp induced by nucleotide ligands, in the absence of drug substrates which may generate complications due to their own binding. Inside-out membrane vesicles containing human Pgp were isolated from multidrug-resistant SKOV/VLB cells and treated with trypsin in the absence or presence of MgATP, Mg-adenosine 5'-[β,γ-imido]triphosphate (Mg-p[NH]ppA) and MgADP. Changes in the proteolysis profile of Pgp owing to binding of nucleotides were used to indicate the conformational changes in Pgp. We found that generation of tryptic fragments, including the loop linking transmembrane (TM) regions TM8 and TM9 of Pgp, were stimulated by the binding of Mg-p[NH]ppA, MgATP and MgADP, indicating that the Pgp conformation was changed by the binding of these nucleotides. The effects of nucleotides on Pgp conformation are directly associated with the binding and/or hydrolysis of these ligands. Four conformational states of Pgp were stabilized under different conditions with various ligands and inhibitors. We propose that cycling through these four states couples the Pgp-mediated MgATP hydrolysis to drug transport.

Original languageEnglish (US)
Pages (from-to)897-904
Number of pages8
JournalBiochemical Journal
Volume328
Issue number3
StatePublished - Dec 15 1997
Externally publishedYes

Fingerprint

P-Glycoprotein
Nucleotides
Adenosine Triphosphate
Proteolysis
Ligands
Conformations
Hydrolysis
Pharmaceutical Preparations
Adenosine Diphosphate
Adenosine Triphosphatases
Membrane Transport Proteins
Multiple Drug Resistance
Adenosine
Trypsin

ASJC Scopus subject areas

  • Biochemistry

Cite this

Wang, G., Pincheira, R., Zhang, M., & Zhang, J-T. (1997). Conformational changes of P-glycoprotein by nucleotide binding. Biochemical Journal, 328(3), 897-904.

Conformational changes of P-glycoprotein by nucleotide binding. / Wang, Guichun; Pincheira, Roxana; Zhang, Mei; Zhang, Jian-Ting.

In: Biochemical Journal, Vol. 328, No. 3, 15.12.1997, p. 897-904.

Research output: Contribution to journalArticle

Wang, G, Pincheira, R, Zhang, M & Zhang, J-T 1997, 'Conformational changes of P-glycoprotein by nucleotide binding', Biochemical Journal, vol. 328, no. 3, pp. 897-904.
Wang G, Pincheira R, Zhang M, Zhang J-T. Conformational changes of P-glycoprotein by nucleotide binding. Biochemical Journal. 1997 Dec 15;328(3):897-904.
Wang, Guichun ; Pincheira, Roxana ; Zhang, Mei ; Zhang, Jian-Ting. / Conformational changes of P-glycoprotein by nucleotide binding. In: Biochemical Journal. 1997 ; Vol. 328, No. 3. pp. 897-904.
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