Biochemical Analysis of Multidrug Resistance-linked Tran

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1.Elucidation of the catalytic cycle of ATP hydrolysis by Pgp and role of conserved motifs in the ATP-binding cassette:We are continuing our studies on the catalytic cycle of ATP hydrolysis by Pgp. Based on the thermodynamic and kinetic properties we have identified the E-S and E-P stable reaction intermediates of the Pgp-mediated ATPase reaction. Using this defined framework and the Walker B E556Q/E1201Q double mutant, we can precisely attribute the high to low affinity switch in the transport substrate binding site to the formation of the E-S reaction intermediate. Importantly, the post-hydrolysis E-P state continues to have low affinity for drug-substrate, suggesting that conformational changes that form the E-S complex are coupled to the transport substrate site to do mechanical work. Thus, the formation of the E-S reaction intermediate during a single turnover of the catalytic cycle appears to provide the initial power stroke for movement of drug substrates from the inner leaflet to the outer leaflet of the lipid bilayer. To complement these studies, the data mining, sequence alignment of 18K ABC domains and site-directed mutagenesis approaches are used to assess the role of critical residues in the conserved subdomains in the NBDs in ATP-binding and hydrolysis. We recently described a conserved subdomain of the ABC, which we named the A-loop (Aromatic residue interacting with the Adenine ring of ATP). This aromatic residue, 25 residues upstream of the Walker A motif, is conserved in 85% of ABC proteins. Biochemical and mutagenesis studies demonstrate that this residue is critical for ATP-binding. Homology modeling of the NBDs of Pgp based on the structure of a dimer of the E171Q mutant MJ0796 NBD shows that the tyrosine (Y401 or Y1044) residue stacks against the adenine base of ATP through ?a-?a interactions. 2.Development of synthetic or natural product modulators that inhibit multiple ABC drug transporters:Work from several labs including ours has established that ABCB1, ABCG2 and ABCC1 are the major contributors to the development of MDR and these transporters exhibit significant overlap in substrate specificity. To develop modulator(s) that will inhibit multiple transporters, we screened synthetic compounds from several sources as well as natural products. We found that curcumin isolated from the turmeric powder, which is consumed daily as a spice in many countries, is a potent modulator of all three transporters. Interestingly, curcumin is not very cytotoxic and it is not transported by ABCB1, C1 or G2. In addition to curcumin, tetrahydroxy curcumin, which is a major metabolite of curcumin, was also found to modulate the function of these transporters (these studies were carried out in collaboration with Dr. P. Limtrakul, Chiang Mai University, Thailand). In addition, in collaboration with Dr. Susan Bates (NCI, NIH) we demonstrated that dihydropyridines, which are used in the clinic to treat hypertension, are transport substrates for ABCG2, similar to ABCB1. Thus, both ABCB1 and G2 might be an important determinant for the potency and use of dihydropyridines for the treatment of hypertension. 3.Resolution of three-dimensional structure of human Pgp:The resolution of the three-dimensional structure of Pgp is an ongoing project in collaboration with Dr. Di Xia in the Laboratory of Cell Biology. Currently, we have been able to obtain 10-12 mg pure Pgp/ml using either 1,2-diheptanoyl-sn-glycero-phosphocholine (DHPC) or dodecyl maltoside detergent. In addition to wild-type protein, several mutants including the E556Q/E1201Q double mutant, which is trapped in an E-S pre-hydrolysis transition-like state in the presence of ATP, have also been purified. Similarly, to stabilize the conformation of wild-type protein in the E-S pre-hydrolysis transition state, we have developed conditions including the use of ATP-??-S, which is a non-hydrolyzable analog of ATP.