P-glycoprotein (Pgp; mouse MDR3) was expressed inPichia pastoris, grown in fermentor culture, and purified. The final pure product is of high specific ATPase activity and is soluble at low detergent concentration. 120 g of cells yielded 6 mg of pure Pgp; >4 kg of cells were obtained from a single fermentor run. Properties of the pure protein were similar to those of previous preparations, except there was significant ATPase activity in absence of added lipid. Mutant mouse MDR3 Pglycoproteins were purified by the same procedure after growth of cells in flask culture, with similar yields and purity. This procedure should open up new avenues of structural, biophysical, and biochemical studies of Pgp. Equilibrium nucleotide-binding parameters of wildtype mouse MDR3 Pgp were studied using 2-(3)-O-(2,4,6-trinitrophenyl)adenosine tri-and diphosphate. Both analogs were found to bind with K d in the low micromolar range, to a single class of site, with no evidence of cooperativity. ATP displacement of the analogs was seen. Similar binding was seen with K429R/K1072R and D551N/D1196N mutant mouse MDR3 Pgp, showing that these Walker A and B mutations had no significant effect on affinity or stoichiometry of nucleotide binding. These residues, known to be critical for catalysis, are concluded to be involved primarily in stabilization of the catalytic transition state in Pgp. P-glycoprotein (Pgp)1 is a plasma membrane-located, ATPdriven drug efflux pump that confers multidrug resistance on mammalian cells (1-5). It occurs commonly in human tumors and is a major obstacle to successful chemotherapy. Consisting of two duplicated "halves" and a total length of around 1280 residues, it is a prominent member of the ABC transporter superfamily (6) and shows the typical ABC transporter domain arrangement consisting of two transmembrane domains (TMD) and two nucleotide-binding sites (NBS), in a linear sequence that can be represented as TMD1-NBS1-TMD2-NBS2. Current studies in many laboratories are aimed at understanding Pgp structure, function, normal physiology, and pharmacology.Our laboratory has studied the two nucleotide sites, and we have established that both are catalytic MgATP hydrolysis sites, which interact together closely (7-10). Earlier, we proposed (11) a catalytic mechanism for Pgp, in which the two NBS hydrolyze MgATP alternately, and hydrolysis is coupled to drug transport from an inner-facing, higher affinity, drug-binding site to an outer-facing, lower affinity site. Subsequent reports (12-16) have supported and extended this proposed mechanism. Further work (17-20) on the structure and location of the drug-binding sites has shown that certain transmembrane ␣-helices are involved in forming these sites, and work (21) with the closely related LmrA protein showed that transport is indeed from the inner lipid leaflet to the outer surface as proposed in Ref. 22. The mechanism of coupling of energy of ATP hydrolysis to transport of drugs is not well understood, however, at the present time. A preliminary structure of Pgp...

It is known from earlier work that two conserved Glu residues, designated "catalytic carboxylates," are critical for function in P-glycoprotein (Pgp). Here the role of these residues (Glu-552 and Glu-1197 in mouse MDR3 Pgp) was studied further. Mutation E552Q or E1197Q reduced Pgp-ATPase to low but still measurable rates. Two explanations previously offered for effects of these mutations, namely that ADP release is slowed or that a second (drug site-resetting) round of ATP hydrolysis is blocked, were evaluated and appeared unsatisfactory. Thus the study was extended to include E552A, -D, and -K and E1197A, -D, and -K mutants. All reduced ATPase to similar low but measurable rates. Orthovanadate-trapping experiments showed that mutation to Gln, Ala, Asp, or Lys altered characteristics of the transition state but did not eliminate its formation in contrast e.g. with mutation of the analogous catalytic Glu in F 1 -ATPase. Retention of ATP as well as ADP was seen in Ala, Asp, and Lys mutants. Mutation E552A in nucleotide binding domain 1 (NBD1) was combined with mutation S528A or S1173A in the LSGGQ sequence of NBD1 or NBD2, respectively. Synergistic effects were seen. E552A/S1173A had extremely low turnover rate for ATPase, while E552A/S528A showed zero or close to zero ATPase. Both showed orthovanadate-independent retention of ATP and ADP. We propose that mutations of the catalytic Glu residues interfere with formation and characteristics of a closed conformation, involving an interdigitated NBD dimer interface, which normally occurs immediately following ATP binding and progresses to the transition state.

Human wild-type and Cys-less P-glycoproteins were expressed in Pichia pastoris and purified in high yield in detergent-soluble form. Both ran on SDS gels as a single 140-kDa band in the presence of reducing agent and showed strong verapamil-stimulated ATPase activity in the presence of added lipid. The wild type showed spontaneous formation of higher molecular mass species in the absence of reducing agent, and its ATPase was activated by dithiothreitol. Oxidation with Cu 2؉ generated the same higher molecular mass species, primarily at 200 and ϳ300 kDa, in high yield. Cross-linking was reversed by dithiothreitol and prevented by pretreatment with N-ethylmaleimide. Using proteins containing different combinations of naturally occurring Cys residues, it was demonstrated that an inhibitory intramolecular disulfide bond forms between Cys-431 and Cys-1074 (located in the Walker A sequences of nucleotide-binding sites 1 and 2, respectively), giving rise to the 200-kDa species. In addition, dimeric P-glycoprotein species (ϳ300 kDa) form by intermolecular disulfide bonding between Cys-431 and Cys-1074. The ready formation of the intramolecular disulfide between Cys-431 and Cys-1074 establishes that the two nucleotide-binding sites of P-glycoprotein are structurally very close and capable of intimate functional interaction, consistent with available information on the catalytic mechanism. Formation of such a disulfide in vivo could, in principle, underlie a regulatory mechanism and might provide a means of intervention to inhibit P-glycoprotein. P-glycoprotein (Pgp)1 is a plasma membrane-located member of the ABC transporter family, which hydrolyzes ATP and uses the energy to pump a wide variety of drugs and other hydrophobic compounds out of cells. It is a major contributor to multidrug resistance in mammalian cells and is recognized as an impediment to therapy with anticancer and anti-AIDS drugs (1-6). Pgp consists of ϳ1280 amino acid residues, with the domain structure TMD1-NBS1-TMD2-NBS2, where TMD indicates a membrane domain consisting of six predicted transmembrane helices, and NBS1 and NBS2 indicate N-and Cterminal nucleotide-binding sites of Pgp, respectively, containing Walker A, Walker B, and ABC transporter signature consensus sequences. The drug-binding sites are proposed to be formed from several transmembrane helices in the membrane domains (7-11). ATP hydrolysis in the two nucleotide sites was proposed to occur via an alternating sites mechanism in which formation and collapse of the catalytic transition state are coupled to movement of drug across the membrane from the inner to outer surface (12). Considerable support for this mechanism has come from studies using the transition state analog MgADP-vanadate (13-16) and the ground state analog MgADP-beryllium fluoride (17) and additionally from chemical modification (18,19) and specific mutagenesis of the catalytic sites (19,20). Understanding the structure and mechanism of action of Pgp in detail is fundamental to devising ways to circumvent multidrug resi...

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...