Vinca alkaloid binding to P-glycoprotein occurs in a processive manner

Abstract

A mechanistic understanding of how P-glycoprotein (Pgp) is able to bind and transport its astonishing range of substrates remains elusive. Pharmacological data demonstrated the presence of at least four distinct binding sites, but their locations have not been fully elucidated. The combination of biochemical and structural data suggests that initial binding may occur in the central cavity or at the lipid-protein interface. Our objective was to define the binding sites for two transported substrates of Pgp; the anticancer drug vinblastine and the fluorescent probe rhodamine 123. A series of mutations was generated in positions proximal to previously defined drug-interacting residues on Pgp. The protein was purified and reconstituted into styrene-maleic acid lipid particles (SMALPs) to measure the apparent drug binding constant or into liposomes for assessment of drug-stimulated ATP hydrolysis. The biochemical data were reconciled with structural models of Pgp using molecular docking. The data indicated that the binding of rhodamine 123 occurred predominantly within the central cavity of Pgp. In contrast, the significantly more hydrophobic vinblastine bound to both the lipid-protein interface and within the central cavity. The data suggest that the initial interaction of vinca alkaloids with Pgp occurs at the lipid interface followed by internalisation into the central cavity, which also provides the transport conduit. This model is supported by recent structural observations with Pgp and early biophysical and cross-linking approaches. Moreover, the proposed model illustrates that the broad substrate profile for Pgp is underpinned by a combination of multiple initial interaction sites and an accommodating transport conduit.

Publication DOI: https://doi.org/10.1016/j.bbamem.2022.184005
Divisions: College of Health & Life Sciences > School of Biosciences
Funding Information: The authors thank the Australian National University and the Research School of Biology for providing scholarships to S. Iqbal and R. Skrzypek and block grant funding for the project.
Additional Information: © 2022 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license 4.0
Uncontrolled Keywords: ABC protein,ABCB1,Drug binding,Membrane transport,Multidrug resistance,P-glycoprotein,Biophysics,Biochemistry,Cell Biology
Publication ISSN: 1879-2642
Last Modified: 20 May 2024 07:41
Date Deposited: 01 Aug 2022 09:56
Full Text Link:
Related URLs: http://www.scop ... tnerID=8YFLogxK (Scopus URL)
https://www.sci ... 1432?via%3Dihub (Publisher URL)
PURE Output Type: Article
Published Date: 2022-10-01
Published Online Date: 2022-07-19
Accepted Date: 2022-07-12
Authors: Iqbal, Shagufta
Flux, Caitlin
Briggs, Deborah A.
Deplazes, Evelyne
Long, Jiansi
Skrzypek, Ruth
Rothnie, Alice (ORCID Profile 0000-0002-4259-7015)
Kerr, Ian D.
Callaghan, Richard

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