Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.6.1.3 (ATPase)
65,361 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The human multidrug resistance P-glycoprotein (P-gp, ABCB1), a member of the ATP-binding cassette (ABC) family of transport proteins, actively transports many cytotoxic compounds out of the cell. ABC transporters have two nucleotide-binding domains (NBD) and two transmembrane domains. The presence of the conserved "signature" sequence (LSGGQ) in each NBD is a unique feature in these transporters. The function of the signature sequences is unknown. In this study, we tested whether the signature sequences ((531)LSGGQ(535) in NBD1; (1176)LSGGQ(1180) in NBD2) in P-gp are in close proximity to the opposing Walker A consensus nucleotide-binding sequences ((1070)GSSGCGKS(1077) in NBD2; (427)GNSGCGKS(434) in NBD1). Pairs of cysteines were introduced into a Cys-less P-gp at the signature and "Walker A" sites and the mutant P-gps were subjected to oxidative cross-linking. At 4 degrees C, when thermal motion is low, P-gp mutants (L531C(Signature)/C1074(Walker A) and C431(Walker A)/L1176C(Signature) were cross-linked. Cross-linking inhibited the drug-stimulated ATPase activities of these two mutants. Their activities were restored, however, after addition of the reducing agent, dithiothreitol. Vanadate trapping of nucleotide at the ATP-binding sites prevented cross-linking of the mutants. These results indicate that the signature sequences are adjacent to the opposing Walker A site. They likely participate in forming the ATP-binding sites and are displaced upon ATP hydrolysis. The resulting conformational change may be the signal responsible for coupling ATP hydrolysis to drug transport by inducing conformational changes in the transmembrane segments.
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PMID:The "LSGGQ" motif in each nucleotide-binding domain of human P-glycoprotein is adjacent to the opposing walker A sequence. 1222 74

The human multidrug resistance P-glycoprotein (P-gp, ABCB1) uses ATP to transport many structurally diverse compounds out of the cell. It is an ABC transporter with two nucleotide-binding domains (NBDs) and two transmembrane domains (TMDs). Recently, we showed that the "LSGGQ" motif in one NBD ((531)LSGGQ(535) in NBD1; (1176)LSGGQ(1180) in NBD2) is adjacent to the "Walker A" sequence ((1070)GSSGCGKS(1077) in NBD2; (427)GNSGCGKS(434) in NBD1) in the other NBD (Loo, T. W., Bartlett, M. C., and Clarke, D. M. (2002) J. Biol. Chem. 277, 41303-41306). Drug substrates can stimulate or inhibit the ATPase activity of P-gp. Here, we report the effect of drug binding on cross-linking between the LSGGQ signature and Walker A sites (Cys(431)(NBD1)/C1176C(NBD2) and Cys(1074)(NBD2)/L531C(NBD1), respectively). Seven drug substrates (calcein-AM, demecolcine, cis(Z)-flupentixol, verapamil, cyclosporin A, Hoechst 33342, and trans(E)-flupentixol) were tested for their effect on oxidative cross-linking. Substrates that stimulated the ATPase activity of P-gp (calcein-AM, demecolcine, cis(Z)-flupentixol, and verapamil) increased the rate of cross-linking between Cys(431)(NBD1-Walker A)/C1176C(NBD2-LSGGQ) and between Cys(1074)(NBD2-Walker A)/L531C(NBD1-LSGGQ) when compared with cross-linking in the absence of drug substrate. By contrast, substrates that inhibited ATPase activity (cyclosporin A, Hoechst 33342, and trans(E)-flupentixol) decreased the rate of cross-linking. These results indicate that interaction between the LSGGQ motifs and Walker A sites must be essential for coupling drug binding to ATP hydrolysis. Drug binding in the transmembrane domains can induce long range conformational changes in the NBDs, such that compounds that stimulate or inhibit ATPase activity must decrease and increase, respectively, the distance between the Walker A and LSGGQ sequences.
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PMID:Drug binding in human P-glycoprotein causes conformational changes in both nucleotide-binding domains. 1242 6

The human multidrug resistance P-glycoprotein (ABCB1) transports a broad range of structurally diverse compounds out of the cell. The transport cycle involves coupling of drug binding in the transmembrane domains with ATP hydrolysis. Compounds such as verapamil stimulate ATPase activity. We used cysteine-scanning mutagenesis of the transmembrane segments and reaction with the thiol-reactive substrate analog of verapamil, methanethiosulfonate (MTS)-verapamil, to test whether it caused permanent activation of ATP hydrolysis. Here we report that one mutant, I306C(TM5) showed increased ATPase activity (8-fold higher than untreated) when treated with MTS-verapamil and isolated by nickel-chelate chromatography. Drug substrates that either enhance (calcein acetoxymethyl ester, demecolcine, and vinblastine) or inhibit (cyclosporin A and trans-(E)-flupentixol) ATPase activity of Cys-less or untreated mutant I306C P-glycoprotein did not affect the activity of MTS-verapamil-treated mutant I306C. Addition of dithiothreitol released the covalently attached verapamil, and ATPase activity returned to basal levels. Pretreatment with substrates such as cyclosporin A, demecolcine, verapamil, vinblastine, or colchicine prevented activation of mutant I306C by MTS-verapamil. The results suggest that MTS-verapamil reacts with I306C in a common drug-binding site. Covalent modification of I306C affects the long range linkage between the drug-binding site and the distal ATP-binding sites. This results in the permanent activation of ATP hydrolysis in the absence of transport. Trapping mutant I306C in a permanently activated state indicates that Ile-306 may be part of the signal to switch on ATP hydrolysis when the drug-binding site is occupied.
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PMID:Permanent activation of the human P-glycoprotein by covalent modification of a residue in the drug-binding site. 1271 2

LmrA is an ATP binding cassette (ABC) multidrug transporter in Lactococcus lactis that is a structural and functional homologue of the human multidrug resistance P-glycoprotein MDR1 (ABCB1). LmrA is also homologous to MsbA, an essential ABC transporter in Escherichia coli involved in the trafficking of lipids, including Lipid A. We have compared the substrate specificities of LmrA and MsbA in detail. Surprisingly, LmrA was able to functionally substitute for a temperature-sensitive mutant MsbA in E. coli WD2 at non-permissive temperatures, suggesting that LmrA could transport Lipid A. LmrA also exhibited a Lipid A-stimulated, vanadate-sensitive ATPase activity. Reciprocally, the expression of MsbA conferred multidrug resistance on E. coli. Similar to LmrA, MsbA interacted with photoactivatable substrate [3H]azidopine, displayed a daunomycin, vinblastine, and Hoechst 33342-stimulated vanadate-sensitive ATPase activity, and mediated the transport of ethidium from cells and Hoechst 33342 in proteoliposomes containing purified and functionally reconstituted protein. Taken together, these data demonstrate that MsbA and LmrA have overlapping substrate specificities. Our observations imply the presence of structural elements in the recently published crystal structures of MsbA in E. coli and Vibrio cholera (Chang, G., and Roth, C. B. (2001) Science 293, 1793-1800; Chang, G. (2003) J. Mol. Biol. 330, 419-430) that support drug-protein interactions and suggest a possible role for LmrA in lipid trafficking in L. lactis.
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PMID:The ATP binding cassette multidrug transporter LmrA and lipid transporter MsbA have overlapping substrate specificities. 1284 82

The human multidrug resistance P-glycoprotein (P-gp, ABCB1) transports a wide variety of structurally diverse compounds out of the cell. The drug-binding pocket of P-gp is located in the transmembrane domains. Although occupation of the drug-binding pocket by one molecule is sufficient to activate the ATPase activity of P-gp, the drug-binding pocket may be large enough to accommodate two different substrates at the same time. In this study, we used cysteine-scanning mutagenesis to test whether P-gp could simultaneously interact with the thiol-reactive drug substrate, Tris-(2-maleimidoethyl)amine (TMEA) and a second drug substrate. TMEA is a cross-linker substrate of P-gp that allowed us to test for stimulation of cross-linking by a second substrate such as calcein-acetoxymethyl ester, colchicine, demecolcine, cyclosporin A, rhodamine B, progesterone, and verapamil. We report that verapamil induced TMEA cross-linking of mutant F343C(TM6)/V982C(TM12). By contrast, no cross-linked product was detected in mutants F343C(TM6), V982C(TM12), or F343C(TM6)/V982C(TM12) in the presence of TMEA alone. The verapamil-stimulated ATPase activity of mutant F343C(TM6)/V982C(TM12) in the presence of TMEA decreased with increased cross-linking of the mutant protein. These results show that binding of verapamil must induce changes in the drug-binding pocket (induced-fit mechanism) resulting in exposure of residues F343C(TM6)/V982C(TM12) to TMEA. The results also indicate that the common drug-binding pocket in P-gp is large enough to accommodate both verapamil and TMEA simultaneously and suggests that the substrates must occupy different regions in the common drug-binding pocket.
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PMID:Simultaneous binding of two different drugs in the binding pocket of the human multidrug resistance P-glycoprotein. 1290 21

The human multidrug resistance P-glycoprotein (P-gp, ABCB1) actively extrudes a broad range of potentially cytotoxic compounds out of the cell. Key steps in understanding the transport process are binding of drug substrates in the transmembrane domains, initiation of ATPase activity, and subsequent drug efflux. We used cysteine-scanning mutagenesis of the transmembrane segment residues and reaction with the thiol-reactive drug substrate analog of rhodamine, methane-thiosulfonate-rhodamine (MTS-rhodamine), to test whether P-gp could be trapped in an activated state with high levels of ATPase activity. The presence of such an activated P-gp could be used to further investigate P-gp-drug substrate interactions. Single cysteine mutants (149) were treated with MTS-rhodamine, and ATPase activities were determined after removal of unreacted MTS-rhodamine. One mutant, F343C(TM6), showed a 5.8-fold increase in activity after reaction with MTS-rhodamine. Pre-treatment of mutant F343C with rhodamine B protected it from activation by MTS-rhodamine, indicating that residue Cys-343 contributes to the rhodamine-binding site. The ATPase activity of MTS-rhodamine-treated mutant F343C, however, was not stimulated further by colchicine or calcein-AM. By contrast, verapamil and Hoechst 33342 stimulated and inhibited, respectively, the ATPase activity of the MTS-rhodamine-treated mutant F343C. These results indicate that the MTS-rhodamine binding site overlaps that of colchicine and calcein-AM but not that of verapamil and Hoechst 33342 within the common drug-binding pocket.
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PMID:Methanethiosulfonate derivatives of rhodamine and verapamil activate human P-glycoprotein at different sites. 1452 74

ATPase activity associated with P-glycoprotein (Pgp) is characterized by three drug-dependent phases: basal (no drug), drug-activated, and drug-inhibited. To understand the communication between drug-binding sites and ATP hydrolytic sites, we performed steady-state thermodynamic analyses of ATP hydrolysis in the presence and absence of transport substrates. We used purified human Pgp (ABCB1, MDR1) expressed in Saccharomyces cerevisiae (Figler, R. A., Omote, H., Nakamoto, R. K., and Al-Shawi, M. K. (2000) Arch. Biochem. Biophys. 376, 34-46) as well as Chinese hamster Pgp (PGP1). Between 23 and 35 degrees C, we obtained linear Arrhenius relationships for the turnover rate of hydrolysis of saturating MgATP in the presence of saturating drug concentrations (kcat), from which we calculated the intrinsic enthalpic, entropic, and free energy terms for the rate-limiting transition states. Linearity of the Arrhenius plots indicated that the same rate-limiting step was being measured over the temperature range employed. Using linear free energy analysis, two distinct transition states were found: one associated with uncoupled basal activity and the other with coupled drug transport activity. We concluded that basal ATPase activity associated with Pgp is not a consequence of transport of an endogenous lipid or other endogenous substrates. Rather, it is an intrinsic mechanistic property of the enzyme. We also found that rapidly transported substrates bound tighter to the transition state and required fewer conformational alterations by the enzyme to achieve the coupling transition state. The overall rate-limiting step of Pgp during transport is a carrier reorientation step. Furthermore, Pgp is optimized to transport drugs out of cells at high rates at the expense of coupling efficiency. The drug inhibition phase was associated with low affinity drug-binding sites. These results are consistent with an expanded version of the alternating catalytic site drug transport model (Senior, A. E., Al-Shawi, M. K., and Urbatsch, I. L. (1995) FEBS Lett. 377, 285-289). A new kinetic model of drug transport is presented.
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PMID:Transition state analysis of the coupling of drug transport to ATP hydrolysis by P-glycoprotein. 1455 Dec 17

P-glycoprotein (P-gp; ABCB1) transports a wide variety of structurally diverse compounds out of the cell. The protein has two homologous halves joined by a linker region. Each half consists of a transmembrane (TM) domain with six TM segments and a nucleotide-binding domain. The drug substrate-binding pocket is at the interface between the TM segments in each half of the protein. Preliminary studies suggested that the arrangement of the two halves of P-gp shows rotational symmetry (i.e. "head-to-tail" arrangement). Here, we tested this model by determining whether the cytoplasmic ends of TM2 and TM3 in the N-terminal half are in close contact with TM11 in the C-terminal half. Mutants containing a pair of cysteines in TM2/TM11 or TM3/TM11 were subjected to oxidative cross-linking with copper phenanthroline. Two of the 110 TM2/TM11 mutants, V133C(TM2)/G939C(TM11) and C137C(TM2)/A935C (TM11), were cross-linked at 4 degrees C, when thermal motion is reduced. Cross-linking was specific since no cross-linked product was detected in the 100 double Cys TM3/TM11 mutants. Vanadate trapping of nucleotide or the presence of some drug substrates inhibited cross-linking of mutants V133C(TM2)/G939C(TM11) and C137C(TM2)/A935C(TM11). Cross-linking of TM2 and TM11 also blocked drug-stimulated ATPase activity. The close proximity of TM2/TM11 and TM5/TM8 (Loo, T. W., Bartlett, M. C., and Clarke, D. M. (2004) J. Biol. Chem. 279, 7692-7697) indicates that these regions between the two halves must enclose the drug-binding pocket at the cytoplasmic side of P-gp. They may form the "hinges" required for conformational changes during the transport cycle.
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PMID:Val133 and Cys137 in transmembrane segment 2 are close to Arg935 and Gly939 in transmembrane segment 11 of human P-glycoprotein. 1474 22

A glycine 185 to valine mutation of human P-glycoprotein (ABCB1, MDR1) has been previously isolated from high colchicine resistance cell lines. We have employed purified and reconstituted P-glycoproteins expressed in Saccharomyces cerevisiae [Figler et al. (2000) Arch. Biochem. Biophys. 376, 34-46] and devised a set of thermodynamic analyses to reveal the mechanism of improved resistance. Purified G185V enzyme shows altered basal ATPase activity but a strong stimulation of colchicine- and etoposide-dependent activities, suggesting a tight regulation of ATPase activity by these drugs. The mutant enzyme has a higher apparent K(m) for colchicine and a lower K(m) for etoposide than that of wild type. Kinetic constants for other transported drugs were not significantly modified by this mutation. Systematic thermodynamic analyses indicate that the G185V enzyme has modified thermodynamic properties of colchicine- and etoposide-dependent activities. To improve the rate of colchicine or etoposide transport, the G185V enzyme has lowered the Arrhenius activation energy of the transport rate-limiting step. The high transition state energies of wild-type P-glycoprotein, when transporting etoposide or colchicine, increase the probability of nonproductive degradation of the transition state without transport. G185V P-glycoprotein transports etoposide or colchicine in an energetically more efficient way with decreased enthalpic and entropic components of the activation energy. Our new data fully reconcile the apparently conflicting results of previous studies. EPR analysis of the spin-labeled G185C enzyme in a cysteine-less background and kinetic parameters of the G185C enzyme indicate that position 185 is surrounded by other residues and is volume sensitive. These results and atomic detail structural modeling suggest that residue 185 is a pivotal point in transmitting conformational changes between the catalytic sites and the colchicine drug binding domain. Replacement of this residue with a bulky valine alters this communication and results in more efficient transport of etoposide or colchicine.
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PMID:Improved energy coupling of human P-glycoprotein by the glycine 185 to valine mutation. 1504 99

Tyrosine kinase inhibitors (TKIs) are promising new agents for specific inhibition of malignant cell growth and metastasis formation. Because most of the TKIs have to reach an intracellular target, specific membrane transporters may significantly modulate their effectiveness. In addition, the hydrophobic TKIs may interact with so-called multidrug transporters and thus alter the cellular distribution of unrelated pharmacological agents. In the present work, we show that certain TKIs, already in the clinical phase of drug development, directly interact with the ABCG2 multidrug transporter protein with a high affinity. We found that in several in vitro assay systems, STI-571 (Gleevec; imatinib mesylate), ZD1839 (Iressa; gefitinib), and N-[4-[(3-bromophenyl)amino]-6-quinazolinyl]-2-butynamide (EKI-785) interacted with ABCG2 at submicromolar concentrations, whereas other multidrug transporters, human multidrug resistance protein (P-glycoprotein, ABCB1) and human multidrug resistance protein 1 (ABCC1), showed much lower reactivity toward these agents. Low concentrations of the TKIs examined selectively modulated ABCG2-ATPase activity, inhibited ABCG2-dependent active drug extrusion, and significantly affected drug resistance patterns in cells expressing ABCG2. Our results indicate that multidrug resistance protein modulation by TKIs may be an important factor in the clinical treatment of cancer patients. These data also raise the possibility that an extrusion of TKIs by multidrug transporters, e.g., ABCG2, may be involved in tumor cell TKI resistance.
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PMID:High-affinity interaction of tyrosine kinase inhibitors with the ABCG2 multidrug transporter. 1515 41


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