Conserved Walker A Ser Residues in the Catalytic Sites of P-glycoprotein Are Critical for Catalysis and Involved Primarily at the Transition State Step

Abstract

P-glycoprotein mutants S430A/T and S1073A/T, affecting conserved Walker A Ser residues, were characterized to elucidate molecular roles of the Ser and functioning of the two P-glycoprotein catalytic sites. P-glycoprotein (Pgp, 1 also known as multidrug resistance protein) is a mammalian, plasma membrane-located protein of around 1280 amino acid residues, which has the ability to exclude and extrude a wide range of hydrophobic compounds from cells using the energy of ATP hydrolysis. It has particular relevance to use of chemotherapeutic drugs in cancer, and of protease inhibitor drugs for AIDS therapy, because it is able to prevent accumulation of many of these drugs in cells, thus conferring a multidrug-resistant phenotype (1-5). Consequently, there is currently a great deal of interest in development of clinically applicable methods to disable or circumvent Pgp in conjunction with drug therapies.Pgp is a member of the ABC transporter superfamily (6). It contains two transmembrane domains, each consisting of six transmembrane helices (7,8) (23) or mutagenesis (24, 25) was sufficient to prevent even a single turnover of ATP hydrolysis at the other, intact site. These experiments supported a working model of the catalytic mechanism in which the two sites alternate to hydrolyze ATP (26). Further work using vanadate as a photocleavage agent (27) supported the model. Additionally, experiments in which drug binding was assessed using photoaffinity labeling by drug analogs (28 -30) has supported the idea, introduced in Ref. 26, that changes in affinity at the drug binding site(s) are linked to formation and collapse of the catalytic transition state.A detailed understanding, in molecular terms, of the structure of Pgp and the mechanism by which it hydrolyzes ATP and couples this process to drug transport, will lead to advances in overcoming multidrug resistance. No high resolution structure of Pgp is yet available, although in recent work we have described a method for large scale purification of detergent-soluble Pgp (31) that provides sufficient material for crystallization trials. Until high resolution data on Pgp are available, it seems reasonable to utilize the HisP x-ray structure (32) as a guide. HisP is the catalytic, ATP-hydrolyzing subunit of the bacterial ABC transporter, histidine permease (33). It shows significant sequence homology to the two Pgp ATP-binding sites, containing Walker A, Walker B, and ABC signature ("LSGGQ") sequences. In addition, HisP contains a Tyr residue (Tyr-16) which, in the x-ray structure, is stacked against the adenine ring of bound ATP. Tyr-16 corresponds in sequence to each of the two Tyr residues (Tyr-397 and Tyr-1040) 2 that are covalently labeled by the photoaffinity label 8-azido-ADP trapped in the N-and C-terminal ATP-binding sites of Pgp (34). Therefore, significant similarities in structure between the HisP and Pgp catalytic sites are evident. * This work was supported by National Institutes of Health Grant GM50156 (to A. E. S.). The costs of publication of this article...

Combined Mutation of Catalytic Glutamate Residues in the Two Nucleotide Binding Domains of P-glycoprotein Generates a Conformation That Binds ATP and ADP Tightly

“…Data with wild-type Pgp preincubated with V i and MgATP were included as a control, and gave a t1 ⁄2 for release of V i -trapped nucleotide of 70 min (Fig. 4A), consistent with previous results (21,31). The mutants were preincubated without V i , and the calculated t1 ⁄2 values and dissociation rates were as follows: E552A/E1197A, 17.6 min, k ϭ 6.6 ϫ 10 Ϫ4 s Ϫ1 ; E552Q/ E1197Q, 2.5 min, k ϭ 4.6 ϫ 10 Ϫ3 s Ϫ1 .…”

“…We used purified protein activated with lipids and DTT (8) and the natural ligands ATP and ADP to characterize the mutant proteins. Mutant proteins were expressed and purified from fermentergrown P. pastoris cells, as described previously (8,21). They displayed a single band when 5-10 g was run on SDS-gels (not shown) and were obtained in yields equal to or slightly higher than wild-type Pgp.…”

“…Strains of P. pastoris expressing the mutant Pgp were grown in fermenter culture and purified as described (8,21). The Pgp concentration was determined as described previously (31).…”

“…A mutagenesis study (21) led to the proposal that cooperativity between the Pgp NBDs involved formation of a "closed" conformation in which "the two domains must come together to allow residues from one site to project into the other." It was suggested that the closed conformation was required after initial ATP binding to attain the transition state.…”

Structures of P-glycoprotein reveal its conformational flexibility and an epitope on the nucleotide-binding domain

“…Purified P-gp was reduced with 1 mM DTT for 30 min on ice, and then excess DTT was removed by passage through 1-mL Sephadex G-50 centrifuge columns equilibrated in 20 mM Hepes at pH 7.4, 10% glycerol, 250 mM NaCl, and 0.1% DDM as described in ref. 34. Protein was activated with 1% (wt/vol) E. coli polar lipids (Avanti) for 10 min at room temperature.…”