ABCMdb

ABCB1 p.Lys1076Met

Predicted by SNAP2:	A: D (91%), C: D (91%), D: D (95%), E: D (95%), F: D (95%), G: D (95%), H: D (91%), I: D (95%), L: D (95%), M: D (91%), N: D (95%), P: D (95%), Q: D (91%), R: D (91%), S: D (91%), T: D (91%), V: D (91%), W: D (95%), Y: D (95%),
Predicted by PROVEAN:	A: D, C: D, D: D, E: D, F: D, G: D, H: D, I: D, L: D, M: D, N: D, P: D, Q: D, R: D, S: D, T: D, V: D, W: D, Y: D,

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[hide] P-glycoprotein inhibits caspase-8 activation but n... Cell Death Differ. 2002 Nov;9(11):1266-72. Ruefli AA, Tainton KM, Darcy PK, Smyth MJ, Johnstone RW
P-glycoprotein inhibits caspase-8 activation but not formation of the death inducing signal complex (disc) following Fas ligation.
Cell Death Differ. 2002 Nov;9(11):1266-72., [PMID:12404126]

Abstract [show]
Previous studies by our laboratory have shown that the drug transporter protein P-glycoprotein, P-gp, can specifically inhibit Fas-induced caspase-3 activation and apoptosis. Importantly, inhibition of both caspase-3 activation and cell death could be reversed by pharmacological and antibody inhibitors of P-gp function. However, the molecular mechanisms underpinning P-gp-mediated resistance to Fas-induced cell death and caspase activation remained unknown. We therefore sought to identify the point(s) within the death receptor pathway at which P-gp exerted its inhibitory effect and to determine whether the ATPase activity of P-gp was required. Structure-function analysis determined that ATP hydrolysis was necessary for P-gp to confer resistance to Fas-induced caspase activation and cell death. Importantly, although both FADD and caspase-8 were recruited to the Death Inducing Signal Complex (DISC) in wild-type P-gp expressing cells following Fas ligation, subsequent activation of caspase-8 at the DISC was inhibited. The ability of P-gp to inhibit caspase-8 activation was also ATP dependent. These studies demonstrate that P-gp inhibits Fas-induced caspase-8 activation but not formation of the DISC and that this activity of P-gp is dependent on ATP hydrolysis.

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109 The mutant P-gp used in the following studies contained double lysine to alanine substitutions at amino acids 233 (K433M) and 1076 (K1076M) within the Walker A motifs, which disrupted nucleotide binding, and ATPase and drug-efflux activity.4,16 Mutant constructs containing double lysine to alanine substitutions at 433(K433M) and 1076(K1076M) amino acids were generated by excising 4.1 kB Eag1 ± Eag1 fragment from clone pUCFVXMDH3 ± Sal(DAUG)-MDR/Neo (provided by Igor Roninson, Chicago University, Chicago, IL) and cloned into MSCV vector. Login to comment

[hide] Mutational analysis of P-glycoprotein: suppression... Cell Death Differ. 2004 Sep;11(9):1028-37. Tainton KM, Smyth MJ, Jackson JT, Tanner JE, Cerruti L, Jane SM, Darcy PK, Johnstone RW
Mutational analysis of P-glycoprotein: suppression of caspase activation in the absence of ATP-dependent drug efflux.
Cell Death Differ. 2004 Sep;11(9):1028-37., [PMID:15131592]

Abstract [show]
P-glycoprotein (P-gp) can induce multidrug resistance (MDR) through the ATP-dependent efflux of chemotherapeutic agents. We have previously shown that P-gp can inhibit nondrug apoptotic stimuli by suppressing the activation of caspases. To determine if this additional activity is functionally linked to ATP hydrolysis, we expressed wild-type and ATPase-mutant P-gp and showed that cells expressing mutant P-gp could not efflux chemotherapeutic drugs but remained relatively resistant to apoptosis. CEM lymphoma cells expressing mutant P-gp treated with vincristine showed a decrease in the fraction of cells with apoptotic morphology, cytochrome c release from the mitochondria and suppression of caspase activation, yet still accumulated in mitosis and showed a loss of clonogenic potential. The loss of clonogenicity in vincristine-treated cells expressing mutant P-gp was associated with accumulation of cells in mitosis and the presence of multinucleated cells consistent with mitotic catastrophe. The antiapoptotic effect of mutant P-gp was not affected by antibodies that inhibit the efflux function of the protein. These data are consistent with a dual activity model for P-gp-induced MDR involving both ATPase-dependent drug efflux and ATPase-independent inhibition of apoptosis. The structure-function analyses described herein provide novel insight into the mechanisms of action of P-gp in mediating MDR.

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23 As shown in Figure 1 (top panel), cells transduced with MSCV alone (CEM-GFP), MSCV-MDR1 (CEM-P-gpWT ) and two clones transduced with MSCV- MDR1K433M/K1076M (CEM-P-gpMM-3 and CEM-P-gpMM-32 ) were obtained and expressed equivalent levels of intracellular GFP. Login to comment
26 To confirm that the desired form of P-gp was expressed, the regions encoding K433M and K1076M substitutions were amplified by PCR using cDNA obtained from CEM-P-gpWT , CEM-P-gpMM-3 and CEM-P-gpMM-32 cells and sequenced (data not shown). Login to comment
164 Production of retrovirally transduced cell lines Production of MSCV-MDR1 (wild type) and MSCV-MDR1 (K433 M, K1076M) has been previously described.41 These constructs or MSCV alone were transfected into 293T cells with PEQ and RD114 helper viruses by CaPO4 À precipitation. Login to comment
171 Primers for K1076M were: (forward) ATCCCAGTGCTTCAGGGA and (reverse) CCTTATTCCAAGCGGCTT. Login to comment

[hide] Rapid purification of human P-glycoprotein mutants... J Biol Chem. 1995 Sep 15;270(37):21449-52. Loo TW, Clarke DM
Rapid purification of human P-glycoprotein mutants expressed transiently in HEK 293 cells by nickel-chelate chromatography and characterization of their drug-stimulated ATPase activities.
J Biol Chem. 1995 Sep 15;270(37):21449-52., 1995-09-15 [PMID:7665554]

Abstract [show]
P-glycoprotein containing 10 tandem histidine residues at the COOH end of the molecule was transiently expressed in HEK 293 cells and purified by nickel-chelate chromatography. The purified protein had an apparent mass of 170 kDa, and its verapamil-stimulated ATPase activity in the presence of phospholipid was 1.2 mumol/min/mg of P-glycoprotein. We then characterized P-glycoprotein mutants that exhibited altered drug-resistant phenotypes and analyzed the contribution of the two nucleotide binding folds to drug-stimulated ATPase activity. Mutation of residues in either nucleotide binding fold abolished drug-stimulated ATPase activity. The pattern of drug-stimulated ATPase activities of mutants, which conferred increased relative resistance to colchicine (G141V, G185V, G830V) or decreased relative resistance to all drugs (F978A), correlated with their drug-resistant phenotypes. By contrast, the ATPase activity of mutant F335A was significantly higher than that of wild-type enzyme when assayed in the presence of verapamil (3.4-fold), colchicine (9.1-fold), or vinblastine (3.7-fold), even though it conferred little resistance to vinblastine in transfected cells. These results suggest that both nucleotide-binding domains must be intact to couple drug binding to ATPase activity and that the drug-stimulated ATPase activity profile of a mutant does not always correlate with its drug-resistant phenotype.

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95 To determine the contribution of either nucleotide-binding domain to drug-stimulatable ATPase activity, mutations were made to the core amino acids (G432S, K433M, G1075S, and K1076M, respectively) of the homology A consensus sequences. Login to comment
123 Mutants K433M, G1075S, and K1076M had no detectable ATPase activities and are omitted for clarity. Login to comment

[hide] Drug efflux mediated by the human multidrug resist... J Cell Sci. 1994 Dec;107 ( Pt 12):3281-90. Sardini A, Mintenig GM, Valverde MA, Sepulveda FV, Gill DR, Hyde SC, Higgins CF, McNaughton PA
Drug efflux mediated by the human multidrug resistance P-glycoprotein is inhibited by cell swelling.
J Cell Sci. 1994 Dec;107 ( Pt 12):3281-90., [PMID:7706385]

Abstract [show]
P-glycoprotein (P-gp), the product of the human multidrug resistance (MDR1) gene, confers multidrug resistance on cells by acting as an ATP-dependent drug transporter. A method using confocal microscopy was developed to measure the transport activity of P-gp from the rate of movement of doxorubicin, a fluorescent substrate of P-gp, across the membrane of a single cell. Recent work has shown that expression of P-gp enhances the activation of chloride channels in response to cell swelling, suggesting that membrane stretch might switch P-gp from a drug-transporting mode to a mode in which it activates chloride channels. In agreement with this idea, we find that cell swelling inhibits drug efflux in cells expressing P-gp but is without effect on the slower background efflux in cells not expressing P-gp and in cells transiently transfected with a mutated MDR1 in which the ATP hydrolysis sites had been inactivated. The identification of a novel means for inhibiting P-gp-mediated drug transport may have implications for the reversal of multidrug resistance during chemotherapy.

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36 Plasmids, mutagenesis and transient expression system Plasmids pMDR7, expressing the human MDR1 gene, and pMDR712, expressing the human MDR1 gene mutated in both ATP binding domains (K433M and K1076M), were constructed as previously described (Valverde et al., 1992; Gill et al., 1992). Login to comment

[hide] Altered drug-stimulated ATPase activity in mutants... J Biol Chem. 1996 Jan 26;271(4):1877-83. Muller M, Bakos E, Welker E, Varadi A, Germann UA, Gottesman MM, Morse BS, Roninson IB, Sarkadi B
Altered drug-stimulated ATPase activity in mutants of the human multidrug resistance protein.
J Biol Chem. 1996 Jan 26;271(4):1877-83., 1996-01-26 [PMID:8567633]

Abstract [show]
The characteristics of P-glycoprotein (MDR1), an ATP-dependent drug extrusion pump responsible for the multidrug resistance of human cancer, were investigated in an in vitro expression system. The wild-type and several mutants of the human MDR1 cDNA were engineered into recombinant baculoviruses and the mutant proteins were expressed in Sf9 insect cells. In isolated cell membrane preparations of the virus-infected cells the MDR1-dependent drug-stimulated ATPase activity, and 8-azido-ATP binding to the MDR1 protein were studied. We found that when lysines 433 and/or 1076 were replaced by methionines in the ATP-binding domains, all these mutations abolished drug-stimulated ATPase activity independent of the MgATP concentrations applied. Photoaffinity labeling with 8-azido-ATP showed that the double lysine mutant had a decreased ATP-binding affinity. In the MDR1 mutant containing a Gly185 to Val replacement we found no significant alteration in the maximum activity of the MDR1-ATPase or in its activation by verapamil and vinblastine, and this mutation did not modify the MgATP affinity or the 8-azido-ATP binding of the transporter either. However, the Gly185 to Val mutation significantly increased the stimulation of the MDR1-ATPase by colchicine and etoposide, while slightly decreasing its stimulation by vincristine. These shifts closely correspond to the effects of this mutation on the drug-resistance profile, as observed in tumor cells. These data indicate that the Sf9-baculovirus expression system for MDR1 provides an efficient tool for examining structure-function relationships and molecular characteristics of this clinically important enzyme.

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38 In the present experiments we used site-directed mutagenesis to alter single amino acids of the human Pgp in the homology A consensus sequences in the NBDs of the NH2-terminal (Lys433 to Met) and/or COOH-terminal (Lys1076 to Met) halves, and applied the cDNA of the spontaneous Gly185 to Val substitution mutant. Login to comment

[hide] Protein kinase C-mediated phosphorylation does not... J Biol Chem. 1996 Jun 7;271(23):13668-74. Goodfellow HR, Sardini A, Ruetz S, Callaghan R, Gros P, McNaughton PA, Higgins CF
Protein kinase C-mediated phosphorylation does not regulate drug transport by the human multidrug resistance P-glycoprotein.
J Biol Chem. 1996 Jun 7;271(23):13668-74., 1996-06-07 [PMID:8662768]

Abstract [show]
P-glycoprotein (P-gp) is an active transporter that can confer multidrug resistance by pumping cytotoxic drugs out of cells and tumors. P-gp is phosphorylated at several sites in the "linker" region, which separates the two halves of the molecule. To examine the role of phosphorylation in drug transport, we mutated P-gp such that it could no longer be phosphorylated by protein kinase C (PKC). When expressed in yeast, the ability of the mutant proteins to confer drug resistance, or to mediate [3H]vinblastine accumulation in secretory vesicles, was indistinguishable from that of wild type P-gp. A matched pair of mammalian cell lines were generated expressing wild type P-gp and a non-phosphorylatable mutant protein. Mutation of the phosphorylation sites did not alter P-gp expression or its subcellular localization. The transport properties of the mutant and wild type proteins were indistinguishable. Thus, phosphorylation of the linker of P-gp by PKC does not affect the rate of drug transport. In light of these data, the use of agents that alter PKC activity to reverse multidrug resistance in the clinic should be considered with caution.

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27 The construction of pMDR712, expressing the human MDR1 gene mutated in both ATP-binding domains (K433M and K1076M), has been described previously (Gill et al., 1992). Login to comment

[hide] P-glycoprotein function involves conformational tr... Proc Natl Acad Sci U S A. 1997 Nov 25;94(24):12908-13. Mechetner EB, Schott B, Morse BS, Stein WD, Druley T, Davis KA, Tsuruo T, Roninson IB
P-glycoprotein function involves conformational transitions detectable by differential immunoreactivity.
Proc Natl Acad Sci U S A. 1997 Nov 25;94(24):12908-13., 1997-11-25 [PMID:9371774]

Abstract [show]
The MDR1 P-glycoprotein (Pgp), a member of the ATP-binding cassette family of transporters, is a transmembrane ATPase efflux pump for various lipophilic compounds, including many anti-cancer drugs. mAb UIC2, reactive with the extracellular moiety of Pgp, inhibits Pgp-mediated efflux. UIC2 reactivity with Pgp was increased by the addition of several Pgp-transported compounds or ATP-depleting agents, and by mutational inactivation of both nucleotide-binding domains (NBDs) of Pgp. UIC2 binding to Pgp mutated in both NBDs was unaffected in the presence of Pgp transport substrates or in ATP-depleted cells, whereas the reactivities of the wild-type Pgp and Pgps mutated in a single NBD were increased by these treatments to the level of the double mutant. These results indicate the existence of different Pgp conformations associated with different stages of transport-associated ATP hydrolysis and suggest trapping in a transient conformation as a mechanism for antibody-mediated inhibition of Pgp.

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22 The mutant Pgps contain K433M and͞or K1076M substitutions in the Walker A motifs; in addition, the cloning procedure resulted in a Q1280A substitution of the C-terminal amino acid in all of the transfected MDR1 cDNA sequences. Login to comment

[hide] Mutations in either nucleotide-binding site of P-g... Biochemistry. 1998 Mar 31;37(13):4592-602. Urbatsch IL, Beaudet L, Carrier I, Gros P
Mutations in either nucleotide-binding site of P-glycoprotein (Mdr3) prevent vanadate trapping of nucleotide at both sites.
Biochemistry. 1998 Mar 31;37(13):4592-602., 1998-03-31 [PMID:9521779]

Abstract [show]
Vanadate trapping of nucleotide and site-directed mutagenesis were used to investigate the role of the two nucleotide-binding (NB) sites in the regulation of ATP hydrolysis by P-glycoprotein (mouse Mdr3). Mdr3, tagged with a hexahistidine tail, was overexpressed in the yeast Pichia pastoris and purified to about 90% homogeneity by Ni-affinity chromatography. This protocol yielded purified, reconstituted Mdr3 which exhibited high verapamil stimulation of ATPase activity with a Vmax of 4.2 micromol min-1 mg-1 and a KM of 0.7 mM, suggesting that Mdr3 purified from P. pastoris is highly functional. Point mutations were introduced into the core consensus sequence of the Walker A or B motifs in each of the two NB sites. The mutants K429R, K1072R (Walker A) and D551N, D1196N (Walker B) were functionally impaired and unable to confer cellular resistance to the fungicide FK506 in the yeast Saccharomyces cerevisiae. Single and double mutants (K429R/K1072R, D551N/D1196N) were expressed in P. pastoris, and the effect of these mutations on the ATPase activity of Mdr3 was characterized. Purified reconstituted Mdr3 mutants showed no detectable ATPase activity compared to proteoliposomes purified from negative controls (<5% of wild-type Mdr3). Vanadate readily induced trapping of 8-azido-nucleotide in the wild-type enzyme after a short 10 s incubation, and specific photolabeling of Mdr3 after UV irradiation. No such vanadate-induced trapping/photolabeling was observed in any of the mutants, even after a 60 min trapping period at 37 degrees C. Since vanadate trapping with 8-azido-ATP requires hydrolysis of the nucleotide, the data suggest that 8-azido-ATP hydrolysis is dramatically impaired in all of the mutant proteins (<0.3% activity). These results show that mutations in either NB site prevent single turnover and vanadate trapping of nucleotide in the nonmutant site. These results further suggest that the two NB sites cannot function independently as catalytic sites in the intact molecule. In addition, the N- or C-terminal NB sites appear functionally indistinguishable, and cooperative interactions absolutely required for ATP hydrolysis may originate from both sites.

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29 In purified reconstituted human MDR1, mutations at the corresponding positions (K433M, K1076M) abolished verapamil-stimulated ATPase activity (24). Login to comment

[hide] Drug-stimulated nucleotide trapping in the human m... J Biol Chem. 1998 Apr 24;273(17):10132-8. Szabo K, Welker E, Bakos, Muller M, Roninson I, Varadi A, Sarkadi B
Drug-stimulated nucleotide trapping in the human multidrug transporter MDR1. Cooperation of the nucleotide binding domains.
J Biol Chem. 1998 Apr 24;273(17):10132-8., 1998-04-24 [PMID:9553060]

Abstract [show]
The human multidrug transporter (MDR1 or P-glycoprotein) is an ATP-dependent cellular drug extrusion pump, and its function involves a drug-stimulated, vanadate-inhibited ATPase activity. In the presence of vanadate and MgATP, a nucleotide (ADP) is trapped in MDR1, which alters the drug binding properties of the protein. Here, we demonstrate that the rate of vanadate-dependent nucleotide trapping by MDR1 is significantly stimulated by the transported drug substrates in a concentration-dependent manner closely resembling the drug stimulation of MDR1-ATPase. Non-MDR1 substrates do not modulate, whereas N-ethylmaleimide, a covalent inhibitor of the ATPase activity, eliminates vanadate-dependent nucleotide trapping. A deletion in MDR1 (Delta amino acids 78-97), which alters the substrate stimulation of its ATPase activity, similarly alters the drug dependence of nucleotide trapping. MDR1 variants with mutations of key lysine residues to methionines in the N-terminal or C-terminal nucleotide binding domains (K433M, K1076M, and K433M/K1076M), which bind but do not hydrolyze ATP, do not show nucleotide trapping either with or without the transported drug substrates. These data indicate that vanadate-dependent nucleotide trapping reflects a drug-stimulated partial reaction of ATP hydrolysis by MDR1, which involves the cooperation of the two nucleotide binding domains. The analysis of this drug-dependent partial reaction may significantly help to characterize the substrate recognition and the ATP-dependent transport mechanism of the MDR1 pump protein.

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5 MDR1 variants with mutations of key lysine residues to methionines in the N-terminal or C-terminal nucleotide binding domains (K433M, K1076M, and K433M/K1076M), which bind but do not hydrolyze ATP, do not show nucleotide trapping either with or without the transported drug substrates. Login to comment
39 Baculovirus transfer vectors were constructed as described earlier (20, 21) by using the human MDR1 cDNA encoding a protein with the following mutants: ⌬aa 78-97, K433M, K1076M, and K433M/K1076M. Login to comment
117 These lysines were replaced by methionines either in the N-terminal ABC domain (K433M), in the C-terminal ABC domain (K1076M), or in both ABC domains (K433M/K1076M). Login to comment
125 Nucleotide trapping in the nucleotide binding site mutants of MDR1. Labeling was performed in Sf9 cell membranes expressing either beta-galactosidase, the wild-type human MDR1, or the nucleotide binding site mutants K433M, K1076M, and K433M/ K1076M. Login to comment
135 As shown, there was no significant azido-ATP trapping in either the K433M or K1076M mutant MDR1 proteins, whereas the corresponding immunoblots ensured that the isolated Sf9 cell membranes contained about equal amounts of the MDR1 variants in all experiments. Login to comment
162 The mutant MDR1 proteins, in which lysines in the first (K433M), second (K1076M), or both nucleotide binding domains are replaced by methionines, were demonstrated to bind MgATP less efficiently at low MgATP concentrations (2-5 ␮M) but similarly to the wild-type MDR1 at concentrations above 10 ␮M MgATP (21). Login to comment

[hide] Identification of mutations in the putative ATP-bi... J Clin Invest. 1994 Aug;94(2):516-20. Fanen P, Guidoux S, Sarde CO, Mandel JL, Goossens M, Aubourg P
Identification of mutations in the putative ATP-binding domain of the adrenoleukodystrophy gene.
J Clin Invest. 1994 Aug;94(2):516-20., [PMID:8040304]

Abstract [show]
The recently identified adrenoleukodystrophy (ALD) gene is predicted to encode a peroxisomal protein of 745 amino acids that includes one domain for ATP-binding, termed nucleotide-binding fold (NBF). To determine whether mutations occur in the putative NBF of ALD protein, we analyzed by denaturing gradient gel electrophoresis (DGGE) exon 6 and 8 that encode most part of this domain in 50 ALD patients. Four amino acid substitutions, three frameshift mutations leading to premature termination signal, and a splicing mutation were identified. These amino acid substitutions occurred at residues highly conserved in other ATP-binding cassette (ABC) proteins. In addition, a nonsense mutation was detected in exon 4.

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138 The chloride channel activity of the CFTR mutant S 1255P, which involves the same amino acid position in the NBF as in the ALD mutant R518W, is less sensitive to ATP stimulation (24) and the MDR mutants K433M or K1076M within the same Walker motif are unable to hydrolyze ATP (25). Login to comment

[hide] The power of the pump: mechanisms of action of P-g... Eur J Pharm Sci. 2006 Apr;27(5):392-400. Epub 2005 Dec 13. Ambudkar SV, Kim IW, Sauna ZE
The power of the pump: mechanisms of action of P-glycoprotein (ABCB1).
Eur J Pharm Sci. 2006 Apr;27(5):392-400. Epub 2005 Dec 13., [PMID:16352426]

Abstract [show]
Members of the superfamily of ATP-binding cassette (ABC) transporters mediate the movement of a variety of substrates including simple ions, complex lipids and xenobiotics. At least 18 ABC transport proteins are associated with disease conditions. P-glycoprotein (Pgp, ABCB1) is the archetypical mammalian ABC transport protein and its mechanism of action has received considerable attention. There is strong biochemical evidence that Pgp moves molecular cargo against a concentration gradient using the energy of ATP hydrolysis. However, the molecular details of how the energy of ATP hydrolysis is coupled to transport remain in dispute and it has not been possible to reconcile the data from various laboratories into a single model. The functional unit of Pgp consists of two nucleotide binding domains (NBDs) and two trans-membrane domains which are involved in the transport of drug substrates. Considerable progress has been made in recent years in characterizing these functionally and spatially distinct domains of Pgp. In addition, our understanding of the domains has been augmented by the resolution of structures of several non-mammalian ABC proteins. This review considers: (i) the role of specific conserved amino acids in ATP hydrolysis mediated by Pgp; (ii) emerging insights into the dimensions of the drug binding pocket and the interactions between Pgp and the transport substrates and (iii) our current understanding of the mechanisms of coupling between energy derived from ATP binding and/or hydrolysis and efflux of drug substrates.

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52 Between the Walker A and B sequences is found a linker peptide with the sequence LSGGQ, also known as the C-region or ABC signature sequence, as it is the hallmark of Table 1 - Summary of mutational analysis of conserved residues in nucleotide-binding domains of Pgp Domain Source Residue number Function Reference NBD1 NBD2 A-loop Human Y401A Y1044A No ATP binding/hydrolysis Kim et al. (submitted for publication) Walker A Mouse K429N K1072N Normal ATP binding but no hydrolysis Azzaria et al. (1989) G431A G1073A Human C431 C1074 ATP protects from modification by N-ethylmaleimide Loo and Clarke (1995) Disulfide bond formation between Walker A domains of both NBDs Urbatsch et al. (2001) Human K433M K1076M Decreased ATP-binding Muller et al. (1996) No ATP hydrolysis Szakacs et al. (2000) No vanadate-trapping, but aluminum and beryllium fluoride-induced trapping normal Q-loop Mouse Q471 Q1114 Not essential for ATP hydrolysis but may be involved in communication with drug-substrate sites Urbatsch et al. (2000a) LSGGQ or linker peptide or signature motif Mouse S528A S1173A Normal ATP binding but no hydrolysis Tombline et al. (2004a) Human S532R Decreased cell surface expression Hoof et al. (1994) Human G534C G1179C No ATP hydrolysis Loo et al. (2002) Human G534D Decreased cell surface expression Hoof et al. (1994) No drug resistance Normal cell surface expression Bakos et al. (1997) No ATP hydrolysis Human G534D/V G1179D Interdomain communication Szakacs et al. (2001) Human Q535C Q1180C No ATP hydrolysis Loo et al. (2002) Human K536Q Decreased drug resistance Hoof et al. (1994) LSGGQ or linker peptide or signature motif Human K536R Increased colchicine resistance (normal ATP hydrolysis?) Login to comment

[hide] New light on multidrug binding by an ATP-binding-c... Trends Pharmacol Sci. 2006 Apr;27(4):195-203. Epub 2006 Mar 20. Shilling RA, Venter H, Velamakanni S, Bapna A, Woebking B, Shahi S, van Veen HW
New light on multidrug binding by an ATP-binding-cassette transporter.
Trends Pharmacol Sci. 2006 Apr;27(4):195-203. Epub 2006 Mar 20., [PMID:16545467]

Abstract [show]
ATP-binding-cassette (ABC) multidrug transporters confer multidrug resistance to pathogenic microorganisms and human tumour cells by mediating the extrusion of structurally unrelated chemotherapeutic drugs from the cell. The molecular basis by which ABC multidrug transporters bind and transport drugs is far from clear. Genetic analyses during the past 14 years reveal that the replacement of many individual amino acids in mammalian multidrug resistance P-glycoproteins can affect cellular resistance to drugs, but these studies have failed to identify specific regions in the primary amino acid sequence that are part of a defined drug-binding pocket. The recent publication of an X-ray crystallographic structure of the bacterial P-glycoprotein homologue MsbA and an MsbA-based homology model of human P-glycoprotein creates an opportunity to compare the original mutagenesis data with the three-dimensional structures of transporters. Our comparisons reveal that mutations that alter specificity are present in three-dimensional 'hotspot' regions in the membrane domains of P-glycoprotein.

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58 Although mutation of only one of these residues (L975A, V981A and F983A) has no effect on the phenotype of the protein [20], double mutations either completely inhibit (V981A/F983A and L975A/V981A) or cause 50% inhibition (L975A/F983A) of Table 1. Login to comment
59 Published mutations in human and murine P-glycoprotein that alter drug transport in cells Location of mutation Mutation Refs Mutation Refs Mutation Refs Transmembrane helices H61A and others [14] I214L [60] L868W [59] G64R [15] P223A [65] I936A [21] L65R [15] S224P [60] F938A [21] Q139[H/P/R] [60] I306R [18] S939[A/C/T/Y/W/D/F] [21,22] G141V [17] F335A [16] T941A [21] G185V [61,62] V338A [66] Q942A [21] I186N [61] G338A [67,68] A943G [21] G187V [17] A339P [67,68] Y946A [21] G187E [60] G341A [66] S948A [21] A192T [60] S344[A/T/C/Y] [66] Y949A [21] F200L [60] N350I [19] C952A [21] F204S [60] P709A [65] F953A [21] R206L [60] G830V [17] L975A [20] W208G [60] I837L [23] F978A [16] K209E [60] N839I [23] V981A [20] L210I [60] I862F [19] F983A [20] T211P [60] L865F [19] F978A [16] V213A [60] P866A [65] N988D [59] Intracellular domain T169I [60] K177I [60] G288V [17] R170L [60] E180G [60] A931T [19] L171P [60] G181R [60] F934A [21] T172P [60] G183D [60] G935A [21] S176P [60] D184N [60] NBD D555N [63] K1076M [69] E1197Q [64] D558N [64] D1093N [64] D1203N [64] D592N [64] E1125Q [64] D1237N [64] E604Q [64] S1173A [70] E1249Q [64] Review TRENDS in Pharmacological Sciences Vol.27 No. Login to comment

[hide] P-glycoprotein-mediated colchicine resistance in d... Biochemistry. 2001 Apr 10;40(14):4323-31. Druley TE, Stein WD, Ruth A, Roninson IB
P-glycoprotein-mediated colchicine resistance in different cell lines correlates with the effects of colchicine on P-glycoprotein conformation.
Biochemistry. 2001 Apr 10;40(14):4323-31., [PMID:11284688]

Abstract [show]
The multidrug transporter P-glycoprotein (Pgp) is an ATPase efflux pump for multiple cytotoxic agents, including vinblastine and colchicine. We have found that resistance to vinblastine but not to colchicine in cell lines derived from different types of tissues and expressing the wild-type human Pgp correlates with the Pgp density. Vinblastine induces a conformational change in Pgp, evidenced by increased reactivity with a conformation-sensitive monoclonal antibody UIC2, in all the tested cell lines. In contrast, colchicine increases the UIC2 reactivity in only some of the cell lines. In those lines where colchicine alone did not affect UIC2 reactivity, this drug was, however, able to reverse the vinblastine-induced increase in UIC2 reactivity. The magnitude of the increase in UIC2 reactivity in the presence of saturating concentrations of colchicine correlates with the relative ability of Pgp to confer colchicine resistance in different cell lines, suggesting the existence of some cell-specific factors that have a coordinate effect on the ability of colchicine to induce conformational transitions and to be transported by Pgp. Colchicine, like vinblastine, reverses the decrease in UIC2 reactivity produced by nonhydrolyzable nucleotides, but unlike vinblastine, it does not reverse the effect of ATP at a high concentration. Colchicine, however, decreases the Hill number for the effect of ATP on the UIC2 reactivity from 2 to 1. Colchicine increases the UIC2 reactivity and reverses the effect of ATP in ATPase-deficient Pgp mutants, but not in the wild-type Pgp expressed in the same cellular background, suggesting that ATP hydrolysis counteracts the effects of colchicine on the Pgp conformation.

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38 The MK, KM, and MM mutants contain amino acid substitutions at either one (KM or MK) or both (MM) conserved lysine residues in the Walker A motifs of the N-terminal or C-terminal NBS, K433M and K1076M, respectively. Login to comment
135 Figure 4B compares the effects of increasing concentrations of colchicine on UIC2 reactivity of LMtk- cell lines transfected with either the wild-type human Pgp (KK-H) or Pgp mutants carrying K433M or K1076M substitutions of the essential lysine residues in the Walker A motifs of the N-terminal (MK-H) or C-terminal (KM-H) NBS, respectively. Login to comment

[hide] Analysis of MDR1 P-glycoprotein conformational cha... Biochemistry. 2001 Apr 10;40(14):4312-22. Druley TE, Stein WD, Roninson IB
Analysis of MDR1 P-glycoprotein conformational changes in permeabilized cells using differential immunoreactivity.
Biochemistry. 2001 Apr 10;40(14):4312-22., [PMID:11284687]

Abstract [show]
The reactivity of the ATP-dependent multidrug transporter P-glycoprotein (Pgp) with the conformation-sensitive monoclonal antibody UIC2 is increased in the presence of Pgp transport substrates, ATP-depleting agents, or mutations that reduce the level of nucleotide binding by Pgp. We have investigated the effects of nucleotides and vinblastine, a Pgp transport substrate, on the UIC2 reactivity of Pgp in cells permeabilized by Staphylococcus aureus alpha-toxin. ATP, ADP, and nonhydrolyzable ATP analogues decreased the UIC2 reactivity; this effect was potentiated by vanadate, a nucleotide-trapping agent. The Hill number for the nucleotide-induced conformational transition was 2 for ATP and ADP but 1 for nonhydrolyzable ATP analogues. The Hill numbers for ATP and ADP were decreased to 1 by mutations in one of the two nucleotide binding sites of Pgp, whereas mutation of both sites greatly diminished the overall effect of nucleotides. Vinblastine reversed the decrease in the UIC2 reactivity brought about by all the nucleotides, including nonhydrolyzable analogues; this effect of vinblastine was blocked by vanadate. These data indicate that UIC2-detectable conformational changes of Pgp are driven by binding and debinding of nucleotides, that nucleotide hydrolysis affects the Hill number for its Pgp interactions, and that Pgp transport substrates promote nucleotide dissociation from Pgp. These findings are consistent with a conventional E1/E2 model that explains conformational transitions of a transporter protein through a series of linked equilibria.

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49 Site-directed mutagenesis was used to generate the MK, KM, and MM mutants, which contain amino acid substitutions at either one (KM or MK) or both (MM) conserved lysine residues in the Walker A motifs of the N-terminal or C-terminal nucleotide-binding sites, K433M and K1076M, respectively. Login to comment

[hide] Transition-state formation in ATPase-negative muta... Biochem Biophys Res Commun. 2000 Oct 5;276(3):1314-9. Szakacs G, Ozvegy C, Bakos E, Sarkadi B, Varadi A
Transition-state formation in ATPase-negative mutants of human MDR1 protein.
Biochem Biophys Res Commun. 2000 Oct 5;276(3):1314-9., [PMID:11027628]

Abstract [show]
In this work we have studied the partial catalytic reactions in MDR1 variants carrying mutations in the conserved Walker A region (K433M and K1076M) of either the N-terminal or C-terminal ABC domain. Both mutations have been demonstrated to cause a loss of drug transport, drug-stimulated ATPase, and vanadate-dependent nucleotide trapping activity. Here we show that these mutants still allow transition state formation (nucleotide trapping) when fluoro-aluminate or beryllium fluoride is used as a complex-stabilizing anion. Drug stimulation of nucleotide trapping was found to be preserved in both mutants. Limited trypsin digestion revealed that whenever MDR1-nucleotide trapping occurred, both ABC domains were involved in the formation of the catalytic intermediates. Our results show that details of the MDR1-ATPase cycle can be studied even in ATPase-negative mutants. These data also demonstrate that the conformational alteration caused by a mutation in one of the ABC domains is propagated to the other, nonmutated domain, indicating a tight coupling between the functioning of the two ABC domains.

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0 Transition-State Formation in ATPase-Negative Mutants of Human MDR1 Protein Gergely Szaka´cs,* Csilla O¨ zvegy,† E´ va Bakos,*, † Bala´zs Sarkadi,* and Andra´s Va´radi†,1 *National Institute of Haematology and Immunology, Membrane Research Group, Hungarian Academy of Sciences, Daro´czi ut 24, H-1113 Budapest, Hungary; and †Institute of Enzymology, Biological Research Center, Hungarian Academy of Sciences, Karolina ut 29, H-1113 Budapest, Hungary Received August 26, 2000 In this work we have studied the partial catalytic reactions in MDR1 variants carrying mutations in the conserved Walker A region (K433M and K1076M) of either the N-terminal or C-terminal ABC domain. Login to comment
26 We have shown earlier that the replacement of a lysine residue with methionine in the catalytic Walker A motif in either ABC domains of the human MDR1 (K433M and K1076M) eliminated drug-stimulated ATPase activity, while these mutants could still bind ATP [13]. Login to comment
59 The arrow indicates the position of human MDR1 on the blot. Nucleotide Trapping in the Walker A Mutants in the Presence of AlF4 and BeFx: Effects of Drugs Our earlier results showed that the replacement of lysine with methionine in either of the Nor C-terminal Walker A sequences (mutations K433M and K1076M) resulted in the loss of ATPase activity [13], although these mutations did not interfere with MgATP-binding. Login to comment
78 Labeling was performed by the incubation of 100 ␮g of Sf9 cell membranes expressing K433M MDR1 (lanes 1, 2 and 5, 6) or K1076M MDR1 (lanes 3, 4 and 7, 8), at 37°C for 10 min in the presence of 20 ␮M [␣-32 P]-8-azido-ATP and 2 mM MgCl2, as described under Materials and Methods. Login to comment
82 Labeling was performed by the incubation of 200 ␮g of Sf9 cell membranes expressing wild-type MDR1 (lanes 1 and 2), K433M MDR1 (lanes 3 and 4), K1076M MDR1 (lanes 5 and 6), at 37°C for 10 min in the presence of 20 ␮M [␣-32 P]-8-azido-ATP and 2 mM MgCl2. Login to comment
85 The arrows indicate the position of human MDR1 and the tryptic fragments containing the N-terminal and C-terminal halves, as revealed by immunostaining of the same the blot. by AlF4 are formed in both ABC domains of the wild-type, as well as of the K433M and the K1076M mutant variants (reactions with BeFx gave similar results, not shown). Login to comment
89 We have replaced a key lysine of the Walker A motif (GCGKST in both ABC domains) to methionine (K433M and K1076M). Login to comment
108 Drug stimulation of the rate of formation of the occluded nucleotide transitory complexes was preserved in the Walker A mutants, K433M and K1076M (Fig. 2). Login to comment
109 In the double mutant, K433M/K1076M, no nucleotide trapping was detected, regardless of the choice of the stabilizing anion (not shown). Login to comment
132 However, the results of this study show that a unilateral mutation (K1076M, that prevents the vanadate-dependent labeling, but allows labeling in the presence of AlF4 and BeFx), has the same effect on both sites: the C-terminal mutation results in an altered conformation, and this minor alteration of the mutated active center is "propagated" to the other (native) catalytic site. Login to comment

[hide] Non-equivalent cooperation between the two nucleot... Biochim Biophys Acta. 1998 Aug 14;1373(1):131-6. Takada Y, Yamada K, Taguchi Y, Kino K, Matsuo M, Tucker SJ, Komano T, Amachi T, Ueda K
Non-equivalent cooperation between the two nucleotide-binding folds of P-glycoprotein.
Biochim Biophys Acta. 1998 Aug 14;1373(1):131-6., [PMID:9733949]

Abstract [show]
To identify the roles of the two nucleotide-binding folds (NBFs) in the function of human P-glycoprotein, a multidrug transporter, we mutated the key lysine residues to methionines and the cysteine residues to alanines in the Walker A (WA) motifs (the core consensus sequence) in the NBFs. We examined the effects of these mutations on N-ethylmaleimide (NEM) and ATP binding, as well as on the vanadate-induced nucleotide trapping with 8-azido-[alpha-32P]ATP. Mutation of the WA lysine or NEM binding cysteine in either of the NBFs blocked vanadate-induced nucleotide trapping of P-glycoprotein. These results suggest that if one NBF is non-functional, there is no ATP hydrolysis even if the other functional NBF contains a bound nucleotide, further indicating the strong cooperation between the two NBFs of P-glycoprotein. However, we found that the effect of NEM modification at one NBF on ATP binding at the other NBF was not equivalent, suggesting a non-equivalency of the role of the two NBFs in P-glycoprotein function.

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73 Vanadate-induced nucleotide trapping and ATP binding in K433M and K1076M mutant P-glycoproteins To further examine the roles of the two NBFs in the function of P-glycoprotein, we constructed three other mutants, K433M, K1076M, and K433M/ K1076M, in which the lysine residue in the WA motif of either or both NBFs was replaced by methionine. Login to comment
78 Immunoblot analysis (A), vanadate-induced nucleotide trapping (B), and 8-azido-ATP binding (C) of wild-type protein and lysine-to-methionine mutants of P-glycoprotein-S. Lanes: 1, cells not transfected; 2, P-glycoprotein-S; 3, K433M; 4, K1076M; 5, K433M/K1076M. Login to comment
86 As previously reported all these mutants bound ATP, although the binding to the K433M/K1076M double-mutant form appeared substantially reduced (lane 5). Login to comment

[hide] The catalytic cycle of P-glycoprotein. FEBS Lett. 1995 Dec 27;377(3):285-9. Senior AE, al-Shawi MK, Urbatsch IL
The catalytic cycle of P-glycoprotein.
FEBS Lett. 1995 Dec 27;377(3):285-9., [PMID:8549739]

Abstract [show]
P-glycoprotein is a plasma-membrane glycoprotein which confers multidrug-resistance on cells and displays ATP-driven drug-pumping in vitro. It contains two nucleotide-binding domains, and its structure places it in the 'ABC transporter' family. We review recent evidence that both nucleotide-sites bind and hydrolyse Mg-ATP. The two catalytic sites interact strongly. A minimal scheme for the MgATP hydrolysis reaction is presented. An alternating catalytic sites scheme is proposed, in which drug transport is coupled to relaxation of a high-energy catalytic site conformation generated by the hydrolysis step. Other ABC transporters may show similar catalytic features.

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103 Loo and Clarke [37] extended this approach, showing that the mutations K433M, K1076M, G432S and G1075S in the Homology A sequences of either NBS1 or NBS2 in human Pgp abolish drug-exclusion capability in cells and eliminate ATPase activity in membranes. Login to comment