Abstract
The transport cycle of ABC transporters in general and P-glycoprotein in particular has been extensively studied, but the molecular mechanism remains controversial. We identify stable reaction intermediates in the progression of the P-glycoprotein-mediated ATPase reaction equivalent to the enzyme-substrate (E.S, P-glycoprotein.ATP) and enzyme-product (E.P, P-glycoprotein.ADP.P(i)) reaction intermediates. These have been characterized using the photoaffinity analog 8-azido-[alpha-32P]ATP as well as under equilibrium conditions using [alpha-32P]ATP, in which a cross-linking step is not involved. Similar results were obtained when 8-azido-[alpha-32P]ATP or [alpha-32P]ATP was used. The reaction intermediates were characterized based on their kinetic properties and the nature (triphosphate/diphosphate) of the trapped nucleotide. Using this defined framework and the Walker B E556Q/E1201Q mutant that traps nucleotide in the absence of vanadate or beryllium fluoride, the high to low affinity switch in the transport substrate binding site can be attributed to the formation of the E.S reaction intermediate of the ATPase reaction. Importantly, the posthydrolysis E.P state continues to have low affinity for substrate, suggesting that conformational changes that form the E.S complex are coupled to the conformational change at the transport substrate site to do mechanical work. Thus, the formation of E.S reaction intermediate during a single turnover of the catalytic cycle appears to provide the initial power stroke for movement of drug substrate from inner leaflet to outer leaflet of lipid bilayer. This novel approach applies transition state theory to elucidate the mechanism of P-glycoprotein and other ABC transporters and has wider applications in testing cause-effect hypotheses in coupled systems.
Highlights
The ATP-binding cassette (ABC)2 family of transport proteins is one of the largest families of proteins in living organisms [1, 2]
Using this defined framework and the Walker B E556Q/E1201Q mutant that traps nucleotide in the absence of vanadate or beryllium fluoride, the high to low affinity switch in the transport substrate binding site can be attributed to the formation of the E1⁄7S reaction intermediate of the ATPase reaction
The ABC transport proteins have discrete nucleotide binding domains (NBDs) and transport substrate binding sites [7], and it is generally accepted that transport of various substrates from simple ions to complex toxic compounds and proteins involves coupling the energy of ATP hydrolysis to mechanical movements at the transport substrate sites [3,4,5]
Summary
Chemicals—[125I]IAAP (2,200 Ci/mmol) and [␣-32P]ATP (3,000 Ci/mmol) were obtained from PerkinElmer Life Sciences. 8-Azido-[␣-32P]ATP (15–20 Ci/mmol), 8-azido-[␣32P]ADP (15–20 Ci/mmol), 8-azido-ATP, and 8-azido-ADP were purchased from Affinity Labeling Technologies, Inc. (Lexington, KY). Purification and Reconstitution of Pgp—Human Pgp from crude membranes of High Five insect cells was purified as described previously [9, 23]. Crude membranes of High Five insect cells (100 g) or purified and reconstituted protein (5–10 g) were incubated in the ATP assay buffer (see above) containing 50 M 8-azido[␣-32P]ATP (5 Ci/nmol) and 300 M Vi in the dark at 37 °C for 5 min. The reaction was stopped by the addition of 10 mM ice-cold ATP and placing the samples immediately on ice. The trapped nucleotides were photocross-linked and electrophoresed, and the radioactivity incorporated in the Pgp band was quantified as described [19]. Purified Pgp reconstituted into liposomes (25–50 g of protein/ml) was incubated with 200 M [␣-32P]ATP for 20 min in the ATPase buffer (see above). The procedure adopted to estimate the Eact value for nucleotide occlusion was essentially similar to that adopted by van der Does et al [31]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
