Abstract
ABCB1/P-glycoprotein is an ATP binding cassette transporter that is involved in the clearance of xenobiotics, and it affects the disposition of many drugs in the body. Conformational flexibility of the protein within the membrane is an intrinsic part of its mechanism of action, but this has made structural studies challenging. Here, we have studied different conformations of P-glycoprotein simultaneously in the presence of ivacaftor, a known competitive inhibitor. In order to conduct this, we used high contrast cryo-electron microscopy imaging with a Volta phase plate. We associate the presence of ivacaftor with the appearance of an additional density in one of the conformational states detected. The additional density is in the central aqueous cavity and is associated with a wider separation of the two halves of the transporter in the inward-facing state. Conformational changes to the nucleotide-binding domains are also observed and may help to explain the stimulation of ATPase activity that occurs when transported substrate is bound in many ATP binding cassette transporters.
Highlights
P-glycoprotein (P-gp) is an ATP-dependent multi-drug transporter of the ATP-binding cassette (ABC) family that is involved in the active efflux of many drugs and xenobiotics from cells [1]
P-gp structures have been obtained by X-ray crystallography [14,15,16,17,18] and cryo-electron microscopy [7,8,9,19,20,21]
Ivacaftor stimulated human P-gp ATPase activity eightfold, suggesting it could be considered as a transported allocrite, and its dissociation constant (KD ) was estimated at 0.3 μM using a fluorescent dye transport assay with the purified protein [22]
Summary
P-glycoprotein (P-gp) is an ATP-dependent multi-drug transporter of the ATP-binding cassette (ABC) family that is involved in the active efflux of many drugs and xenobiotics from cells [1]. Drugs and inhibitors are thought to bind to the inward-facing conformation of P-gp [6,7,8,9], which is more favored in the post hydrolytic or nucleotide-free conditions [10,11,12]. The drug is translocated across the plasma membrane by transition of the protein to the outward-facing state, which is more favored when ATP is bound [12]. The dynamic conformational nature of P-gp (and other ABC family members [13]) has challenged structural studies. Stabilizing antibodies and ATP hydrolysis-inactivating mutations have been exploited to generate
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