ABCB3 p.Glu552Gln
| Predicted by SNAP2: | A: D (80%), C: D (75%), D: N (78%), F: D (85%), G: D (85%), H: D (75%), I: D (85%), K: D (85%), L: D (85%), M: D (85%), N: D (80%), P: D (91%), Q: D (75%), R: D (91%), S: D (85%), T: D (85%), V: D (85%), W: D (85%), Y: D (85%), |
| Predicted by PROVEAN: | A: D, C: D, D: N, 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, |
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[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.
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No. Sentence Comment
48 In an effort to gain insight into the mechanism of ATP hydrolysis by P-gp, including testing the role of the two NBDs in catalysis, we have previously mutated the glutamate residues homologous to E179 of HisP in the mouse Mdr3 enzyme (E552Q and E1197Q in NBD1 and NBD2, respectively) (43).
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ABCB3 p.Glu552Gln 14596601:48:235
status: NEW51 In this study, we have attempted to characterize the molecular basis of the defect in the E552Q and E1197Q mutants.
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ABCB3 p.Glu552Gln 14596601:51:90
status: NEW53 The wild-type mouse Mdr3 (WT) and the E552Q, E1197Q, and D551N mutants were created by site-directed mutagenesis and modified by in-frame addition of a six-histidine tag (His6) at the C-terminus of the protein and were expressed in the yeast Pichia pastoris after cloning in the expression plasmid pHIL-D2 (Invitrogen, license 145457), as previously described (43).
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ABCB3 p.Glu552Gln 14596601:53:38
status: NEW108 RESULTS During a previous search for catalytic carboxylate residues in the NBDs of P-glycoprotein two mutants of the mouse Mdr3 isoform at homologous positions in NBD1 and NBD2 (E552Q and E1197Q) showed a similar loss-of-function phenotype, which featured inability to convey multidrug resistance and abrogation of steady-state ATPase activity (43).
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ABCB3 p.Glu552Gln 14596601:108:178
status: NEW110 For this, wild-type (WT) Mdr3 and the E552Q and E1197Q mutants were expressed in the yeast P. pastoris as recombinant proteins bearing an in-frame polyhistidine tail (His6) at the carboxyl terminus.
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ABCB3 p.Glu552Gln 14596601:110:38
status: NEW116 As before, the purified E552Q and E1197Q mutants show a very low basal ATPase activity (0.13-0.18 µmol min-1 mg-1 ) that is not stimulated by drugs (0.13-0.20 µmol min-1 mg-1 ) and which is comparable to the activity seen in the ATPase inactive mutant D551N (49, 51) that is considered to be background.
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ABCB3 p.Glu552Gln 14596601:116:24
status: NEW117 To determine whether the loss of ATPase activity seen in E552Q and E1197Q was caused by an effect of the mutations on affinity for nucleotides, the nucleotide-binding properties of the WT and Mdr3 variants were compared by photoaffinity labeling.
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ABCB3 p.Glu552Gln 14596601:117:57
status: NEW123 Together, these results suggest that the E552Q, E1197Q, and D551N mutations do not have a major effect on nucleotide binding to Mdr3 and are therefore unlikely to cause major nonspecific structural changes in the NBDs.
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ABCB3 p.Glu552Gln 14596601:123:41
status: NEW133 Purified and activated wild-type (WT) and mutant Mdr3 variants (E552Q, E1197Q, D551N) were UV-irradiated on ice in the presence of 3 mM MgCl2 and 5, 10, 20, 40, and 80 µM 8-azido-[R-32P]ATP.
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ABCB3 p.Glu552Gln 14596601:133:64
status: NEW137 Despite the observed lack of ATPase activity of E552Q and E1197Q measured by Pi release, 8-azidonucleotide trapping that is stimulated both by drug and by Vi is readily detectable in these mutants.
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ABCB3 p.Glu552Gln 14596601:137:48
status: NEW140 Although studies of WT Mdr3 [Figure 1 and (43)] and of the inactive mutant D551N [Figure 1 and (43)] suggest that under the hydrolysis conditions used (37 °C; see Experimental Procedures) little if any of the photolabeling is due to 8-azido-[R-32 P]ATP binding, additional experiments were undertaken to verify that the labeling seen in E552Q and E1197Q ((Vi) was due to trapping of hydrolyzed nucleotide, as opposed to simple binding of the label to Mdr3.
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ABCB3 p.Glu552Gln 14596601:140:342
status: NEW141 As formation of the Mg-8-azido-ADP‚Vi complex is optimum at 37 °C and requires Mg2+ ions (28, 53), the temperature dependence and EDTA sensitivity of E552Q and E1197Q photolabeling by 8-azido-[R-32 P]ATP were investigated.
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ABCB3 p.Glu552Gln 14596601:141:162
status: NEW142 Results in Figure 2A,B show that labeling of WT, E552Q, and E1197Q by 8-azido-[R-32 P]ATP under all conditions tested ((Vi, (drugs) was either completely eliminated or largely reduced (>90%) when the incubation and washing steps of the labeling reaction were carried out at 4 °C (Figure 2B) as opposed to 37 °C (Figure 2A).
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ABCB3 p.Glu552Gln 14596601:142:49
status: NEW145 These results are consistent with 8-azido-[R-32 P]ATP hydrolysis in the WT and E552Q and E1197Q mutants with concomitant trapping of 8-azido-[R-32 P]ADP.
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ABCB3 p.Glu552Gln 14596601:145:79
status: NEW152 To obtain further evidence that the E552Q and E1197Q mutants can form the {MgADP‚Vi} transition-state complex, similar nucleotide trapping experiments were carried out in the presence of two other transition-state analogues: aluminum fluoride (AlF4 - ) and beryllium fluoride (BeFx).
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ABCB3 p.Glu552Gln 14596601:152:36
status: NEW157 Results in Figure 3A indicate that AlF4 - can also induce nucleotide trapping in the WT and E552Q and E1197Q mutants, with characteristics similar to those observed when Vi is used to induce trapping (43).
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ABCB3 p.Glu552Gln 14596601:157:92
status: NEW160 These results show that the different transition states revealed by distinct Pi analogues can all be formed in the E552Q and E1197Q mutants in a comparable manner to WT Mdr3.
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ABCB3 p.Glu552Gln 14596601:160:115
status: NEW161 Finally, results in Figure 3C show that BeFx- and AlF4 - -induced nucleotide trapping in the WT and E552Q and E1197Q mutants is EDTA sensitive.
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ABCB3 p.Glu552Gln 14596601:161:100
status: NEW162 Together, these results expand observations with Vi [Figure 3 (43)], showing that in the mutants E552Q and E1197Q activation and cleavage of the bond between the β- and γ-phosphates of ATP seem to take place.
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ABCB3 p.Glu552Gln 14596601:162:97
status: NEW164 As seen in Figure 4, 8-azido- [R-32 P]ADP can be detected following incubation of the WT and E552Q and E1197Q mutants with 8-azido-[R-32 P]ATP and vanadate, while it is absent in the catalytically inactive D551N mutant.
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ABCB3 p.Glu552Gln 14596601:164:93
status: NEW165 Furthermore, formation of ADP does not occur in any of the enzymes when the trapping reaction is carried out at 4 °C. Thus, it appears that the E552Q and E1197Q mutants are able to hydrolyze at least one molecule of ATP.
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ABCB3 p.Glu552Gln 14596601:165:149
status: NEW166 In these experiments, we also detected the presence of ATP in both WT and the E552Q and E1197Q mutants.
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ABCB3 p.Glu552Gln 14596601:166:78
status: NEW182 The nature of the molecular defect in the E552Q and E1197Q mutants was further investigated by comparing the Vi dependence of nucleotide trapping of the WT and the E552Q and E1197Q mutants in a dose-response experiment (0, 0.05 µM e Vi e 100 µM) (Figure 5).
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ABCB3 p.Glu552Gln 14596601:182:42
status: NEWX
ABCB3 p.Glu552Gln 14596601:182:164
status: NEW184 As expected from the results in our previous publication (43), both E552Q and E1197Q could trap 8-azido-[R-32 P]nucleotide at all Vi concentrations tested.
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ABCB3 p.Glu552Gln 14596601:184:68
status: NEW186 E552Q showed low levels of trapping in absence of Vi, but labeling increased in a dose-dependent fashion (similar to the WT enzyme) with very intense labeling seen at 100 µM Vi (>50-fold stimulation).
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ABCB3 p.Glu552Gln 14596601:186:0
status: NEW188 The distinct Vi dose-response behaviors of the two homologous mutants E552Q and E1197Q suggest that NBD1 and NBD2 are not catalytically symmetrical, with NBD2 (intact in E552Q) showing a more robust Vi- dependent trapping than its NBD1 counterpart (intact in E1197Q).
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ABCB3 p.Glu552Gln 14596601:188:70
status: NEWX
ABCB3 p.Glu552Gln 14596601:188:170
status: NEW189 To further investigate a possible functional asymmetry between NBD1 and NBD2, suggested by the Vi dose-response study (Figure 5), we attempted to semiquantitatively assess in which of the NBD(s) of E552Q and E1197Q the nucleotide was trapped, in both the absence and presence of Vi.
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ABCB3 p.Glu552Gln 14596601:189:198
status: NEW194 For E1197Q, results in panels F and H clearly show that all of the label is in the MD7-reactive, C-terminal half of the protein, with little, if any, overlap with the MD13 reactive species detected in panel D. Conversely, and although the overall photolabeling signal is much weaker than that seen for E1197Q, the label incorporated in E552Q colocalizes with the N-terminal and MD13-reactive half (panels D and H) and does not overlap with the faster migrating MD7 positive fragment (panel F).
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ABCB3 p.Glu552Gln 14596601:194:336
status: NEW204 In the presence of Vi, nucleotide trapping was seen in both NBD1 and NBD2 of the E552Q and E1197Q mutants (albeit at different ratios, depending on the position of the mutation).
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ABCB3 p.Glu552Gln 14596601:204:81
status: NEW206 These results indicate that, in WT Mdr3 and the E552Q and E1197Q mutants, both NBDs can hydrolyze at least one FIGURE 6: Trypsin digestion of Mdr3 NB site mutants photolabeled with Mg-8-azido-[R-32P]ATP in the absence of vanadate.
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ABCB3 p.Glu552Gln 14596601:206:48
status: NEW221 Results expressed in the present study suggest that the E552Q and E1197Q mutants are not completely inactive (as opposed to the Walker B mutant D551N) and that these mutants can indeed cleave ATP to ADP and Pi, undergoing partial reactions toward a full cycle of catalysis but never fully turning over.
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ABCB3 p.Glu552Gln 14596601:221:56
status: NEW227 Finally, TLC analysis of the nucleotides bound to the enzymes following Vi trapping of 8-azido-ATP shows formation of 8-azido-ADP by the WT and E552Q and E1197Q mutants (Figure 4).
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ABCB3 p.Glu552Gln 14596601:227:144
status: NEW228 Together, these results indicate that E552Q and E1197Q can indeed cleave ATP to ADP and Pi and that 8-azido-ADP is the nucleotide trapped in the photolabeled enzymes.
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ABCB3 p.Glu552Gln 14596601:228:38
status: NEW230 Therefore, in the E552Q and E1197Q mutants, steps downstream from the formation of the transition state, such as the release of MgADP and/or Pi, or others, must be impaired.
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ABCB3 p.Glu552Gln 14596601:230:18
status: NEW243 The study of E552Q and E1197Q reported here clearly argues in favor of two NBDs that are not functionally equivalent in full-length P-gp.
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ABCB3 p.Glu552Gln 14596601:243:13
status: NEW245 In this experiment it can be observed that E552Q and E1197Q trap nucleotide in the absence of Vi and that the response of each mutant to Vi is completely different, with E552Q showing a strongly dose-dependent increase in labeling (similar to WT), while Vi has little effect on nucleotide trapping in E1197Q.
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ABCB3 p.Glu552Gln 14596601:245:43
status: NEWX
ABCB3 p.Glu552Gln 14596601:245:170
status: NEW247 Since nucleotide trapping in the mutants occurs after cleavage of ATP (with ADP trapped; see above), the differential Vi dose-response observed in E552Q and E1197Q is most easily explained by differential sensitivity of the nonmutant site to inhibition by Vi.
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ABCB3 p.Glu552Gln 14596601:247:147
status: NEW252 In summary, the present work supports the finding that both NBDs are essential for function with complete cooperativity between the two sites and suggests that the E552 and E1197 residues of mouse Mdr3 are probably not the catalytic residues, as the E552Q and E1197Q mutants can hydrolyze ATP in both nucleotide-binding domains.
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ABCB3 p.Glu552Gln 14596601:252:250
status: NEW[hide] New horizon of MDR1 (P-glycoprotein) study. Drug Metab Rev. 2005;37(3):489-510. Mizutani T, Hattori A
New horizon of MDR1 (P-glycoprotein) study.
Drug Metab Rev. 2005;37(3):489-510., [PMID:16257832]
Abstract [show]
MDR1 (once P-glycoprotein, now referred to as ABCB1) plays a role as a blood-brain barrier, preventing drug absorption into the brain, and is known to confer multiple drug resistance in cancer chemotherapy. MDR1 is composed of two repeated fragments, and there are six transmembrane domains (TMD) on the N-terminal of each repeat and a nucleotide (ATP) binding domain (NBD) on the C-terminal. These two repeats are dependent but cooperate as one functional molecule, with one pocket for excreting drugs. The 12 TM domains form a funnel facing the outside of cells, and NBD is in cytosol as a dimer. One NBD is composed of the Walker A, Q-loop, ABC-signature and the Walker B for phosphate binding of nucleotide. This tertiary structure of MDR1 is suggested from the structure of the NBD of histidine permease (HisP), clarified by x-ray crystallography. On the model of HisP, the NBD positions described above make a functional domain, and the same NBD structure is found on many other ABC transporters. An experiment with MDR1 gene knockout mice showed the high plasma AUC of drugs in mdr null mice [mdr1a(-/-)] and a high level in the brain, indicating that MDR1 has an efflux function (prevention of absorption) in the intestinal lumen and acts as a barrier of drug uptake in the brain, as well as has the function of urinary and biliary excretion of drugs. The transcription of MDR1 is dependent on two sites; the promoter site (-105/-100)(-245/-141) and the enhancer site (-7864/-7817). Autoantibody from autoimmune hepatitis patients weakly reacted with the extracellular peptide (aa314-aa328 between TM5 and 6) of MDR1 on the outside of the cell membrane, and did not react with peptides in the NBD and in the membrane-spanning region in TM5. There is an ambiguity about the function of MDR1 as GlcCer translocase.
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No. Sentence Comment
117 E552Q- E1197Q mutation abolished drug-stimulated ATPase activity as measured by release of phosphate, and the results support a model in which two NBDs of MDR1 are not functionally equivalent.
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ABCB3 p.Glu552Gln 16257832:117:0
status: NEW[hide] Catalytic cycle of ATP hydrolysis by P-glycoprotei... Biochemistry. 2007 Dec 4;46(48):13787-99. Epub 2007 Nov 8. Sauna ZE, Kim IW, Nandigama K, Kopp S, Chiba P, Ambudkar SV
Catalytic cycle of ATP hydrolysis by P-glycoprotein: evidence for formation of the E.S reaction intermediate with ATP-gamma-S, a nonhydrolyzable analogue of ATP.
Biochemistry. 2007 Dec 4;46(48):13787-99. Epub 2007 Nov 8., 2007-12-04 [PMID:17988154]
Abstract [show]
Structural and biochemical studies of ATP-binding cassette (ABC) transporters suggest that an ATP-driven dimerization of the nucleotide-binding domains (NBDs) is an important reaction intermediate of the transport cycle. Moreover, an asymmetric occlusion of ATP at one of the two ATP sites of P-glycoprotein (Pgp) may follow the formation of the symmetric dimer. It has also been postulated that ADP drives the dissociation of the dimer. In this study, we show that the E.S conformation of Pgp (previously demonstrated in the E556Q/E1201Q mutant Pgp) can be obtained with the wild-type protein by use of the nonhydrolyzable ATP analogue ATP-gamma-S. ATP-gamma-S is occluded into the Pgp NBDs at 34 degrees C but not at 4 degrees C, whereas ATP is not occluded at either temperature. Using purified Pgp incorporated into proteoliposomes and ATP-gamma-35S, we demonstrate that the occlusion of ATP-gamma-35S has an Eact of 60 kJ/mol and the stoichiometry of ATP-gamma-35S:Pgp is 1:1 (mol/mol). Additionally, in the conserved Walker B mutant (E556Q/E1201Q) of Pgp, we find occlusion of the nucleoside triphosphate but not the nucleoside diphosphate. Furthermore, Pgp in the occluded nucleotide conformation has reduced affinity for transport substrates. These data provide evidence for the ATP-driven dimerization and ADP-driven dissociation of the NBDs, and although two ATP molecules may initiate dimerization, only one is driven to an occluded pre-hydrolysis intermediate state. Thus, in a full-length ABC transporter like Pgp, it is unlikely that there is complete association and disassociation of NBDs and the occluded nucleotide conformation at one of the NBDs provides the power-stroke at the transport-substrate site.
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No. Sentence Comment
261 These studies demonstrated that the E552Q/E1197Q mutant occludes minimal amounts of [14 C]ADP after an extended incubation of 120 min [see Figure 5 in (20)].
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ABCB3 p.Glu552Gln 17988154:261:36
status: NEW[hide] K(ATP) channels "vingt ans apres": ATG to PDB to M... J Mol Cell Cardiol. 2005 Jul;39(1):79-98. Epub 2005 Feb 19. Babenko AP
K(ATP) channels "vingt ans apres": ATG to PDB to Mechanism.
J Mol Cell Cardiol. 2005 Jul;39(1):79-98. Epub 2005 Feb 19., [PMID:15978905]
Abstract [show]
A multidisciplinary effort over twenty years has provided deep insight into the nature of K(ATP) channels. First discovered in cardiomyocytes and pancreatic beta-cells, as ubiquitous sensors of the ADP/ATP ratio they are implicated in multiple disorders characterized by the uncoupling of excitation from metabolism. Composed of two disparate subunits these large octameric channels present a formidable challenge to scientists interested in understanding mechanism in physical, chemical, and structural terms. Post-cloning studies have defined the domains and interactions, within and between the nucleotide-inhibited K(IR) pore and nucleotide-stimulated, drug-binding core of the ATP-Binding Cassette (ABC) regulatory subunits, that control channel assembly and gating. Determination of the three-dimensional structures of the bacterial prototypes of the channel subunits allowed homology modeling and has provided increasingly detailed mechanistic understanding. Here I review the early electrophysiology and molecular biology of K(ATP) channels, cover biophysical principles governing their single channel kinetics, integrate this with current efforts to understand ligand-recognition and gating within the pore and SUR core, and propose a mechanism of coupling based on recent identification of a SUR gatekeeper module and first composite models of (SUR/K(IR) 6.0)(4) complexes. This mechanism, based on interactions between inter-K(IR) subunit ATP-binding pockets and a unique bi-directional regulatory apparatus comprised of elements from the gatekeeper and K(IR) amino terminus, provides a molecular perspective for understanding the biophysical basis underlying the polar effects of pathogenic mutations in K(ATP) channel subunits.
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No. Sentence Comment
265 An E → Q substitution of both NBDs of MDR3 (E552Q/E1197Q) did markedly reduce ATP hydrolysis, while substrate-stimulated, Mg2+ - dependent, vanadate-enhanced photolabeling was retained [145].
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ABCB3 p.Glu552Gln 15978905:265:51
status: NEW