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Research has been conducted on P-glycoprotein, human gene product. The role of conserved glutamate residues in the Walker B domain of the ATP sites has been investigated during human P-glycoprotein catalytic cycle and the details are reported.

Research describes the construction of a combinatorial library of polyenes and their use in the discovery of multidrug resistance reversal polyenes. Results indicate that compounds 4ek and 4ej are the most potent polyenes whose interaction with P-glycoprotein involves both ends of the polyenes.

A genetic screen to isolate second-site mutations of S558Y that restore drug transport was used to characterize the transmission interface in the yeast multidrug transporter Pdr5 that uncouples ATP hydrolysis and drug transport. The results offer insight into the deviant and canonical Q-loop residues which are functionally overlapping and equivalent in a strikingly asymmetric ABC transporter.

The occluded nucleotide conformation in wild-type P-glycoprotein (Pgp) which is generated by using nonhydrolyzable ATP analogue ATP-[gamma]-S is described. These studies could be useful to generate pre-hydrolysis [E.S] intermediate in other ATP-binding cassette (ABC) transporters.

The ATPase activity of the yeast Pdr5p transporter protein by its transport substrate clotrimazole is studied. The results demonstrate that Pdr5p can use GTP, as well as ATP to provide energy for substrate transport, hence increasing its multidrug transport substrate specificity.

A study was conducted to assess the role of the conserved Y401 and Y1044 residues of human P-glycoprotein (Pgp) and these residues were substituted F, W, C, or A either singly or together. Systematic biochemical characterization of these residues demonstrated that only aromatic residues at this position allow full retention of function, called the 'A-loop' of the ABC domain, whereas residues such as L or C support only partial (30-50%) activity.

The dimerization motif GXXXG found in the first transmembrane (TM) of the ABCG2 protein is studied by substituting glycine residues with leucine or alanine. The GXXXG motif facilitates dimerization by allowing packing of the helices though it does not induce dimerization single-handedly.

Human P-glycoprotein (Pgp) is an ATP-dependent efflux pump for a variety of chemotherapeutic drugs. The study is conducted to evaluate whether Pgp modulators can be engineered to exhibit high-affinity binding using polyvalency.

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-γ-S. ATP-γ-S is occluded into the Pgp NBDs at 34 °C but not at 4 °C, whereas ATP is not occluded at either temperature. Using purified Pgp incorporated into proteoliposomes and ATP-γ-35S, we demonstrate that the occlusion of ATP-γ-35S has an Eact of 60 kJ/mol and the stoichiometry of ATP-γ-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. [ABSTRACT FROM AUTHOR]