Catalytic Cycle of ATP Hydrolysis by P-Glycoprotein:  Evidence for Formation of the E·S Reaction Intermediate with ATP-γ-S, a Nonhydrolyzable Analogue of ATP

Abstract

Structural and biochemical studies of ATP-binding cassette (ABC) transporters suggest that an ATP-driven dimerization of the nucleotide-binding domains (NBDs) is an important reaction intermediate of the transport cycle. Moreover, an asymmetric occlusion of ATP at one of the two ATP sites of P-glycoprotein (Pgp) may follow the formation of the symmetric dimer. It has also been postulated that ADP drives the dissociation of the dimer. In this study, we show that the E.S conformation of Pgp (previously demonstrated in the E556Q/E1201Q mutant Pgp) can be obtained with the wild-type protein by use of the nonhydrolyzable ATP analogue ATP-gamma-S. ATP-gamma-S is occluded into the Pgp NBDs at 34 degrees C but not at 4 degrees C, whereas ATP is not occluded at either temperature. Using purified Pgp incorporated into proteoliposomes and ATP-gamma-35S, we demonstrate that the occlusion of ATP-gamma-35S has an Eact of 60 kJ/mol and the stoichiometry of ATP-gamma-35S:Pgp is 1:1 (mol/mol). Additionally, in the conserved Walker B mutant (E556Q/E1201Q) of Pgp, we find occlusion of the nucleoside triphosphate but not the nucleoside diphosphate. Furthermore, Pgp in the occluded nucleotide conformation has reduced affinity for transport substrates. These data provide evidence for the ATP-driven dimerization and ADP-driven dissociation of the NBDs, and although two ATP molecules may initiate dimerization, only one is driven to an occluded pre-hydrolysis intermediate state. Thus, in a full-length ABC transporter like Pgp, it is unlikely that there is complete association and disassociation of NBDs and the occluded nucleotide conformation at one of the NBDs provides the power-stroke at the transport-substrate site.

Human P-glycoprotein Contains a Greasy Ball-and-Socket Joint at the Second Transmission Interface

“…In this "nucleotide-sandwich conforma-tion," each nucleotide would interact with the phosphate-binding loop (P-loop) of one NBD and the ABC signature motif (LSGGQ) of the other NBD. ATP hydrolysis occurs by an alternating site mechanism as inhibition of ATPase activity at one site causes a complete loss of ATPase activity (12)(13)(14)(15).…”

Kinetics of the Association/Dissociation Cycle of an ATP-binding Cassette Nucleotide-binding Domain

“…2) Constant contact model. The NBDs remain in contact during the hydrolysis cycle, and the power stroke results from smaller conformational changes at the NBD-dimer interface (8,(13)(14)(15)17).…”

Dissociation of ATP-binding Cassette Nucleotide-binding Domain Dimers into Monomers during the Hydrolysis Cycle

“…Despite the detailed structural knowledge of the NBDs, the mechanism of nucleotide hydrolysis is controversial. As a result of experimental discrepancies and lack of direct information, a number of models have been proposed (3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14). These models can be broadly divided into the following.…”