Transmembrane helix 12 plays a pivotal role in coupling energy provision and drug binding in ABCB1
Date
2010
Authors
Crowley, Emily
O'Mara, Megan
Kerr, Ian
Callaghan, Richard
Journal Title
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Volume Title
Publisher
Blackwell Publishing Ltd
Abstract
Describing the molecular details of the multidrug efflux process of ABCB1, in particular the interdomain communication associated with bioenergetic coupling, continues to prove difficult. A number of investigations to date have implicated transmembrane helix 12 (TM12) in mediating communication between the transmembrane domains and nucleotide-binding domains (NBDs) of ABCB1. The present investigation further addressed the role of TM12 in ABCB1 by characterizing its topography during the multidrug efflux process with the use of cysteine-directed mutagenesis. Cysteines were introduced at various positions along TM12 and assessed for their ability to covalently bind thiol-reactive fluorescent probes with differing physiochemical properties. By analysing each isoform in the basal, ATP-bound and posthydrolytic states, it was possible to determine how the local environment of TM12 alters during the catalytic cycle. Labelling with hydrophobic CM and zwitterionic BM was extensive throughout the helix in the basal, prehydrolytic and posthydrolytic states, suggesting that TM12 is in a predominantly hydrophobic environment. Overall, the carboxy region (intracellular half) of TM12 appeared to be more responsive to changes in the catalytic state of the protein than the amino region (extracellular half). Thus, the carboxy region of TM12 is suggested to be responsive to nucleotide binding and hydrolysis at the NBDs and therefore directly involved in interdomain communication. This data can be reconciled with an atomic-scale model of human ABCB1. Taken together, these results indicate that TM12 plays a key role in the progression of the ATP hydrolytic cycle in ABCB1 and, in particular, in coordinating conformational changes between the NBDs and transmembrane domains.
Description
Keywords
Keywords: adenosine triphosphate; multidrug resistance protein 1; protein; protein tm12; unclassified drug; article; covalent bond; drug binding; energy; human; priority journal; protein analysis; protein conformation; protein domain; Animals; Binding Sites; Cataly ABC transporter; bioenergetic coupling; drug resistance; efflux pumps; P-glycoprotein
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Source
The FEBS Journal
Type
Journal article
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2037-12-31