ABCB1 p.Glu556Ala
| Predicted by SNAP2: | A: D (85%), C: D (80%), D: N (61%), F: D (91%), G: D (91%), H: D (85%), I: D (85%), K: D (91%), L: D (91%), M: N (72%), N: D (85%), P: D (91%), Q: N (53%), R: D (91%), S: D (85%), T: D (85%), V: D (85%), W: D (91%), Y: D (91%), |
| Predicted by PROVEAN: | A: D, C: D, D: D, F: D, G: D, H: D, I: D, K: D, L: D, M: D, N: D, P: D, Q: D, R: D, S: D, T: D, V: D, W: D, Y: D, |
[switch to compact view]
Comments [show]
None has been submitted yet.
[hide] Importance of the conserved Walker B glutamate res... Biochemistry. 2002 Nov 26;41(47):13989-4000. Sauna ZE, Muller M, Peng XH, Ambudkar SV
Importance of the conserved Walker B glutamate residues, 556 and 1201, for the completion of the catalytic cycle of ATP hydrolysis by human P-glycoprotein (ABCB1).
Biochemistry. 2002 Nov 26;41(47):13989-4000., 2002-11-26 [PMID:12437356]
Abstract [show]
The human MDR1 (ABCB1) gene product, P-glycoprotein (Pgp), functions as an ATP-dependent efflux pump for a variety of chemotherapeutic drugs. In this study, we assessed the role of conserved glutamate residues in the Walker B domain of the two ATP sites (E556 and E1201, respectively) during the catalytic cycle of human Pgp. The mutant Pgps (E556Q, E556A, E1201Q, E1201A, E556/1201Q, and E556/1201A) were characterized using a vaccinia virus based expression system. Although steady-state ATP hydrolysis and drug transport activities were abrogated in both E556Q and E1201Q mutant Pgps, [alpha-(32)P]-8-azidoADP was trapped in the presence of vanadate (Vi), and the release of trapped [alpha-(32)P]-8-azidoADP occurred to a similar extent as in wild-type Pgp. This indicates that these mutations do not affect either the first hydrolysis event or the ADP release step. Similar results were also obtained when Glu residues were replaced with Ala (E556A and E1201A). Following the first hydrolysis event and release of [alpha-(32)P]-8-azidoADP, both E556Q and E1201Q mutant Pgps failed to undergo another cycle of Vi-induced [alpha-(32)P]-8-azidoADP trapping. Interestingly, the double mutants E556/1201Q and E556/1201A trapped [alpha-(32)P]-8-azidoADP even in the absence of Vi, and the occluded nucleotide was not released after incubation at 37 degrees C for an extended period. In addition, the properties of transition state conformation of the double mutants generated in the absence of Vi were found to be similar to that of the wild-type protein trapped in the presence of Vi (Pgp x [alpha-(32)P]-8-azidoADP xVi). Thus, in contrast to the single mutants, the double mutants appear to be defective in the ADP release step. In aggregate, these data suggest that E556 and E1201 residues in the Walker B domains may not be critical as catalytic carboxylates for the cleavage of the bond between the gamma-P and the beta-P of ATP during hydrolysis but are essential for the second ATP hydrolysis step and completion of the catalytic cycle.
Comments [show]
None has been submitted yet.
No. Sentence Comment
2 The mutant Pgps (E556Q, E556A, E1201Q, E1201A, E556/1201Q, and E556/1201A) were characterized using a vaccinia virus based expression system.
X
ABCB1 p.Glu556Ala 12437356:2:24
status: NEW4 Similar results were also obtained when Glu residues were replaced with Ala (E556A and E1201A).
X
ABCB1 p.Glu556Ala 12437356:4:77
status: NEW32 In this study, in addition to the mutants E556Q and E1201Q we have also characterized the E556A and E1201A as well as the double (E556/1201Q and E556/1201A) mutant Pgps.
X
ABCB1 p.Glu556Ala 12437356:32:90
status: NEW54 The coding sequence for the E556Q mutant primer was 5'-ATC CTC CTG CTG GAT CAG GCC ACG TCA GCC TTG-3'; for the E1201Q mutant primer, 5'-ATT TTG CTT TTG GAT CAA GCC ACG TCA GCT CTG-3'; for the E556A mutant primer, 5'-ATC CTC CTG CTG GAT GCG GCC ACG TCA GCC TTG-3'; and for the E1201A primer, 5'-ATT TTG CTT TTG GAT GCA GCC ACG TCA GCT CTG-3'.
X
ABCB1 p.Glu556Ala 12437356:54:192
status: NEW65 Crude membranes were prepared from vTF7-3 infected HeLa cells transfected with vector pTM1-MDR1 wild type, pTM1-MDR1-E556Q, pTM1-MDR1-E1201Q, pTM1-MDR1-E556/1201Q, pTM1-MDR1-E556A, pTM1-MDR1-E1201A, and pTM1-MDR1- E556/1201A as described previously (28, 31) and stored at -70 °C. Total protein was quantified by the Amido Black protein estimation method as previously described (32), and Pgp expression level was determined by immunoblot analysis using the monoclonal antibody C219 (30, 33).
X
ABCB1 p.Glu556Ala 12437356:65:174
status: NEW279 To assess whether a Glu to Ala substitution will affect the cleavage of the -γ-phosphate bond of ATP, we generated the mutant Pgps where Glu was replaced with Ala (E556A, E1201A, and E556/1201A).
X
ABCB1 p.Glu556Ala 12437356:279:170
status: NEW280 Characterization of these mutants showed that (1) they exhibited comparable cell surface expression, (2) similar to E f Q (see Figure 1A), E f A substitutions also resulted in loss of transport activity, and (3) the single mutants E556A and E1201A, similar to E556Q and E1201Q (see Figure 1C), showed some trapping of [R-32 P]-8-azidoADP in the absence of Vi, which was enhanced in the presence of 0.25 mM Vi (data not shown).
X
ABCB1 p.Glu556Ala 12437356:280:231
status: NEW294 Human Pgp mutants of the conserved glutamate residue in the Walker B region (E556Q, E556A, E1201Q, E1201A, E556/1201Q, and E556/1201A) showed cell surface expression levels comparable to that of the wild-type protein, but the transport function was abrogated in all of the mutant Pgps (Figure 1A,B; data for E556A, E1201A, and E556/1201A not shown).
X
ABCB1 p.Glu556Ala 12437356:294:84
status: NEWX
ABCB1 p.Glu556Ala 12437356:294:308
status: NEW333 The single mutants (E556Q, E556A, E1201Q, and E1201A), on the other hand, show normal release of ADP and can bind ATP during next step but exhibit greatly reduced ability to hydrolyze it (see Figure 2A, panels I-III; data with E556A and E1201A not shown).
X
ABCB1 p.Glu556Ala 12437356:333:27
status: NEWX
ABCB1 p.Glu556Ala 12437356:333:227
status: NEW[hide] P-glycoprotein: from genomics to mechanism. Oncogene. 2003 Oct 20;22(47):7468-85. Ambudkar SV, Kimchi-Sarfaty C, Sauna ZE, Gottesman MM
P-glycoprotein: from genomics to mechanism.
Oncogene. 2003 Oct 20;22(47):7468-85., 2003-10-20 [PMID:14576852]
Abstract [show]
Resistance to chemically different natural product anti-cancer drugs (multidrug resistance, or MDR) results from decreased drug accumulation, resulting from expression of one or more ATP-dependent efflux pumps. The first of these to be identified was P-glycoprotein (P-gp), the product of the human MDR1 gene, localized to chromosome 7q21. P-gp is a member of the large ATP-binding cassette (ABC) family of proteins. Although its crystallographic 3-D structure is yet to be determined, sequence analysis and comparison to other ABC family members suggest a structure consisting of two transmembrane (TM) domains, each with six TM segments, and two nucleotide-binding domains. In the epithelial cells of the gastrointestinal tract, liver, and kidney, and capillaries of the brain, testes, and ovaries, P-gp acts as a barrier to the uptake of xenobiotics, and promotes their excretion in the bile and urine. Polymorphisms in the MDR1 gene may affect the pharmacokinetics of many commonly used drugs, including anticancer drugs. Substrate recognition of many different drugs occurs within the TM domains in multiple-overlapping binding sites. We have proposed a model for how ATP energizes transfer of substrates from these binding sites on P-gp to the outside of the cell, which accounts for the apparent stoichiometry of two ATPs hydrolysed per molecule of drug transported. Understanding of the biology, genetics, and biochemistry of P-gp promises to improve the treatment of cancer and explain the pharmacokinetics of many commonly used drugs.
Comments [show]
None has been submitted yet.
No. Sentence Comment
276 Adapted from Sauna et al. (2001a, b) Furthermore, the equivalent mutations in human P-gp, E556Q, E556A, E1201Q, E1201A, and the double mutants E556Q/E1201Q and E556A/E1201A, all allow for normal levels of Vi-dependent [a-32 P]8-azidoADP trapping, and the trapped nucleotide has been demonstrated to be [a-32 P]8-azidoADP (Sauna et al., 2002).
X
ABCB1 p.Glu556Ala 14576852:276:98
status: NEWX
ABCB1 p.Glu556Ala 14576852:276:161
status: NEW277 Thus, the substitutions of the residues E556 and E1201 with Q or A in human P-gp do not block hydrolysis of ATP per se. Interestingly, the double mutants E556Q/ E1201Q and E556A/E1201A show trapping of [a-32 P]8-azidoADP even in the absence of Vi.
X
ABCB1 p.Glu556Ala 14576852:277:172
status: NEW279 These double mutants of P-gps (E556Q/E1201Q and E556A/E1201A) thus provide an independent validation that the Vi-trapped transition state of P-gp is indeed a 'true` transition state, and they provide an interesting system where one can obtain the transition state of P-gp in the absence of agents such as Vi or beryllium fluoride.
X
ABCB1 p.Glu556Ala 14576852:279:48
status: NEW326 The single mutants (E556Q, E556A, E1201Q, and E1201A) on the other hand show normal release of ADP, and can bind ATP during next step, but are unable to hydrolyse it.
X
ABCB1 p.Glu556Ala 14576852:326:27
status: NEW[hide] Analysis of catalytic carboxylate mutants E552Q an... Biochemistry. 2003 Nov 11;42(44):12875-85. Carrier I, Julien M, Gros P
Analysis of catalytic carboxylate mutants E552Q and E1197Q suggests asymmetric ATP hydrolysis by the two nucleotide-binding domains of P-glycoprotein.
Biochemistry. 2003 Nov 11;42(44):12875-85., 2003-11-11 [PMID:14596601]
Abstract [show]
In the nucleotide-binding domains (NBDs) of ABC transporters, such as mouse Mdr3 P-glycoprotein (P-gp), an invariant carboxylate residue (E552 in NBD1; E1197 in NBD2) immediately follows the Walker B motif (hyd(4)DE/D). Removal of the negative charge in mutants E552Q and E1197Q abolishes drug-stimulated ATPase activity measured by P(i) release. Surprisingly, drug-stimulated trapping of 8-azido-[alpha-(32)P]ATP is still observed in the mutants in both the presence and absence of the transition-state analogue vanadate (V(i)), and ADP can be recovered from the trapped enzymes. The E552Q and E1197Q mutants show characteristics similar to those of the wild-type (WT) enzyme with respect to 8-azido-[alpha-(32)P]ATP binding and 8-azido-[alpha-(32)P]nucleotide trapping, with the latter being both Mg(2+) and temperature dependent. Importantly, drug-stimulated nucleotide trapping in E552Q is stimulated by V(i) and resembles the WT enzyme, while it is almost completely V(i) insensitive in E1197Q. Similar nucleotide trapping properties are observed when aluminum fluoride or beryllium fluoride is used as an alternate transition-state analogue. Partial proteolytic cleavage of photolabeled enzymes indicates that, in the absence of V(i), nucleotide trapping occurs exclusively at the mutant NBD, whereas in the presence of V(i), nucleotide trapping occurs at both NBDs. Together, these results suggest that there is single-site turnover occurring in the E552Q and E1197Q mutants and that ADP release from the mutant site, or another catalytic step, is impaired in these mutants. Furthermore, our results support a model in which the two NBDs of P-gp are not functionally equivalent.
Comments [show]
None has been submitted yet.
No. Sentence Comment
229 Moreover, in their recent work, Sauna and colleagues also demonstrated using Rand γ-labeled 8-azido-[R-32 P]ATP that mutants at the equivalent positions of the human MDR1 protein (E556Q and E556A, E1201Q and E1201A, and the double mutants) are indeed capable of ATP hydrolysis and single-site catalysis (59).
X
ABCB1 p.Glu556Ala 14596601:229:196
status: NEW234 Indeed, they suggest that the single E556A/Q and E1202A/Q mutants are defective in the second ATP hydrolysis event of the catalytic cycle, which should reset the protein after hydrolysis at the first site.
X
ABCB1 p.Glu556Ala 14596601:234:37
status: NEW[hide] Combined mutation of catalytic glutamate residues ... J Biol Chem. 2004 Jul 23;279(30):31212-20. Epub 2004 May 24. Tombline G, Bartholomew LA, Urbatsch IL, Senior AE
Combined mutation of catalytic glutamate residues in the two nucleotide binding domains of P-glycoprotein generates a conformation that binds ATP and ADP tightly.
J Biol Chem. 2004 Jul 23;279(30):31212-20. Epub 2004 May 24., 2004-07-23 [PMID:15159388]
Abstract [show]
Combined mutation of "catalytic carboxylates" in both nucleotide binding domains (NBDs) of P-glycoprotein generates a conformation capable of tight binding of 8-azido-ADP (Sauna, Z. E., Muller, M., Peng, X. H., and Ambudkar, S. V. (2002) Biochemistry 41, 13989-14000). Here we characterized this conformation using pure mouse MDR3 P-glycoprotein and natural MgATP and MgADP. Mutants E552A/E1197A, E552Q/E1197Q, E552D/E1197D, and E552K/E1197K had low but real ATPase activity in the order Ala > Gln > Asp > Lys, emphasizing the requirement for Glu stereochemistry. Mutant E552A/E1197A bound MgATP and MgADP (1 mol/mol) with K(d) 9.2 and 92 microm, showed strong temperature sensitivity of MgATP binding and equal dissociation rates for MgATP and MgADP. With MgATP as the added ligand, 80% of bound nucleotide was in the form of ATP. None of these parameters was vanadate-sensitive. The other mutants showed lower stoichiometry of MgATP and MgADP binding, in the order Ala > Gln > Asp > Lys. We conclude that the E552A/E1197A mutation arrests the enzyme in a conformation, likely a stabilized NBD dimer, which occludes nucleotide, shows preferential binding of ATP, does not progress to a normal vanadate-sensitive transition state, but hydrolyzes ATP and releases ADP slowly. Impairment of turnover is primarily due to inability to form the normal transition state rather than to slow ADP release. The Gln, Asp, and Lys mutants are less effective at stabilizing the occluded nucleotide, putative dimeric NBD, conformation. We envisage that in wild-type the occluded nucleotide conformation occurs transiently after MgATP binds to both NBDs with associated dimerization, and before progression to the transition state.
Comments [show]
None has been submitted yet.
No. Sentence Comment
48 Two such mutants have been reported, namely the E556Q/E1201Q and E556A/E1201A mutants of human Pgp (28).
X
ABCB1 p.Glu556Ala 15159388:48:65
status: NEW[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.
Comments [show]
None has been submitted yet.
No. Sentence Comment
53 Hoof et al. (1994) Human L531R Decreased cell surface expression Bakos et al. (1997) G534V K536I Normal cell surface expression K536R Normal ATP hydrolysis I541T R543S LSGGQ or linker peptide or signature motif Human R538M Normal cell surface expression Decreased ATP hydrolysis Bakos et al. (1997) I541R Normal cell surface expression No ATP hydrolysis Walker B Mouse D551N D1196N No ATP hydrolysis, required for Mg2+ binding Urbatsch et al. (1998) Human D555A D1200A Same as above Hrycyna et al. (1999) Walker B Mouse E552A E1197A Trapping of ATP, no steady-state hydrolysis Tombline et al. (2004b) Mouse E552Q E1197Q No steady-state ATP hydrolysis Vigano et al. (2002) Human E556A E1201A Trapping of ATP or ADP in the absence of vanadate, low levels of ATP hydrolysis Sauna et al. (2002) D-loop Mouse D558N D1203N Decreased ATP hydrolysis Urbatsch et al. (2000b) the ABC transporter superfamily.
X
ABCB1 p.Glu556Ala 16352426:53:678
status: NEW