ABCB1 p.Gln1118Ala
| Predicted by SNAP2: | A: D (80%), C: D (85%), D: D (91%), E: D (85%), F: D (91%), G: D (91%), H: D (85%), I: D (91%), K: D (91%), L: D (91%), M: D (80%), N: D (91%), P: D (91%), R: D (91%), S: D (85%), T: D (85%), V: D (85%), W: D (91%), Y: D (85%), |
| Predicted by PROVEAN: | A: D, C: D, D: D, E: D, F: D, G: D, H: D, I: D, K: D, L: D, M: D, N: D, P: D, R: D, S: D, T: D, V: D, W: D, Y: D, |
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[hide] Molecular model and ATPase activity of carboxyl-te... Biochemistry (Mosc). 2006;71 Suppl 1:S18-24, 1-2. Qian F, Wei D, Liu J, Yang S
Molecular model and ATPase activity of carboxyl-terminal nucleotide binding domain from human P-glycoprotein.
Biochemistry (Mosc). 2006;71 Suppl 1:S18-24, 1-2., [PMID:16487063]
Abstract [show]
ATP binding and hydrolysis are required for P-glycoprotein mediated multidrug resistance. To investigate the molecular mechanism involved in ATP binding and hydrolysis, a three-dimensional model of the carboxyl-terminal nucleotide binding domain (NBD2) was built by homology modeling. Modeling revealed the human P-glycoprotein ATP-binding site and the possible role of conserved Gln1118 residue. Recombinant NBD2 was overexpressed in Escherichia coli and the conserved Gln1118 residue was mutated to an alanine residue. The Vmax for ATP hydrolysis by the mutant NBD2 was approximately 56% of the Vmax of wild-type NBD2. But both proteins displayed similar affinity for ATP, with Km of 479 and 466 microM for mutant and wild-type NBD2, respectively. These results suggest that the possible role of Gln1118 is as an activating residue for ATP hydrolysis. The molecular model also provided structural information about the interactions between NBD2 and the chemosensitizer quercetin. The complex indicated that quercetin was tightly bound to the ATP-binding site and competed for binding. The three-dimensional model of NBD2 can be used to both guide enzymological studies and provide a theoretical basis for the design of potential multidrug resistance reversers.
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No. Sentence Comment
12 The functional role of Gln1118 was investigated by the studying the biochem ical properties of recombinant NBD2 and its mutant Q1118A.
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ABCB1 p.Gln1118Ala 16487063:12:127
status: NEW45 Construction of the expression plasmid encoding NBD2 and its mutant Q1118A.
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ABCB1 p.Gln1118Ala 16487063:45:68
status: NEW51 For site directed muta genesis to introduce a Q1118A mutation, nucleotide mutations at positions 3352 (C→G) and 3353 (A→C) were achieved by sequence overlap PCR methodology.
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ABCB1 p.Gln1118Ala 16487063:51:46
status: NEW54 The sequence of plasmid containing NBD2 Q1118A mutant was confirmed by sequencing.
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ABCB1 p.Gln1118Ala 16487063:54:40
status: NEW55 Expression, renaturation, and purification of NBD2 and its mutant Q1118A.
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ABCB1 p.Gln1118Ala 16487063:55:66
status: NEW56 Escherichia coli BL21 cells con taining NBD2 and its mutant Q1118A were cultured in 200 ml of 2YTA medium (16 g/liter tryptone, 10 g/liter yeast extract, 5 g/liter NaCl, and 100 µg/ml ampicillin) at 37°C with vigorous shaking until the absorbance at 600 nm reached 1.2 units.
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ABCB1 p.Gln1118Ala 16487063:56:60
status: NEW124 Expression and purification of NBD2 and its mutant Q1118A.
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ABCB1 p.Gln1118Ala 16487063:124:51
status: NEW127 The Q1118A mutation was introduced into the NBD2 sequence by site directed mutagenesis.
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ABCB1 p.Gln1118Ala 16487063:127:4
status: NEW128 Recombinant proteins encoded by pGEX4T 1 NBD2 and its mutant Q1118A based plas mids were expressed in E. coli cells.
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ABCB1 p.Gln1118Ala 16487063:128:61
status: NEW129 Expression and purification of the NBD2 and its mutant Q1118A are illustrated in Fig. 6.
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ABCB1 p.Gln1118Ala 16487063:129:55
status: NEW135 NBD2 protein and mutant Q1118A protein yielded equal purities and in similar amounts.
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ABCB1 p.Gln1118Ala 16487063:135:24
status: NEW140 Expression and purification of the NBD2 and its mutant Q1118A.
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ABCB1 p.Gln1118Ala 16487063:140:55
status: NEW142 Expression of mutant Q1118A protein before (lane ) and after (lane 4) IPTG induction.
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ABCB1 p.Gln1118Ala 16487063:142:21
status: NEW143 Purified NBD2 protein (lane 5) and its mutant Q1118A protein (lane 6).
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ABCB1 p.Gln1118Ala 16487063:143:46
status: NEW144 S22 QIAN et al. BIOCHEMISTRY (Moscow) Vol. 71 Suppl. 1 2006 ATPase activity of NBD2 and its mutant Q1118A.
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ABCB1 p.Gln1118Ala 16487063:144:99
status: NEW148 The ATPase activities of the Q1118A mutant and that of the wild type NBD2 were compared.
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ABCB1 p.Gln1118Ala 16487063:148:29
status: NEW165 The Q1118A mutant was affected in its ATPase activity but this residue might not participate the binding of ATP because the mutation had no influence on the affinity for ATP.
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ABCB1 p.Gln1118Ala 16487063:165:4
status: NEW[hide] Mini review on molecular modeling of P-glycoprotei... Curr Top Med Chem. 2007;7(15):1525-9. Ha SN, Hochman J, Sheridan RP
Mini review on molecular modeling of P-glycoprotein (Pgp).
Curr Top Med Chem. 2007;7(15):1525-9., [PMID:17897039]
Abstract [show]
Membrane bound P-glycoprotein (Pgp) acts as an active transport pump. It plays a major role as a cause of multidrug resistance (MDR) and acts as a component of the blood-brain barrier. Pgp transports a wide variety of structurally unrelated compound from the cell interior into the extracellular space. Recent molecular modeling efforts, mostly in homology modeling and QSAR studies, have brought some understanding to the interactions between the protein and the drugs at the atomic level. We review the recent developments from the point of view of methodology.
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No. Sentence Comment
61 Also, it is reported that mutation of Gln1118 to Ala reduces hydrolysis of ATP.
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ABCB1 p.Gln1118Ala 17897039:61:38
status: NEW[hide] The Q loops of the human multidrug resistance tran... FASEB J. 2014 Oct;28(10):4335-46. doi: 10.1096/fj.13-245639. Epub 2014 Jul 11. Zolnerciks JK, Akkaya BG, Snippe M, Chiba P, Seelig A, Linton KJ
The Q loops of the human multidrug resistance transporter ABCB1 are necessary to couple drug binding to the ATP catalytic cycle.
FASEB J. 2014 Oct;28(10):4335-46. doi: 10.1096/fj.13-245639. Epub 2014 Jul 11., [PMID:25016028]
Abstract [show]
For a primary active pump, such as the human ATP-binding-cassette (ABC) transporter ABCB1, coupling of drug-binding by the two transmembrane domains (TMDs) to the ATP catalytic cycle of the two nucleotide-binding domains (NBDs) is fundamental to the transport mechanism, but is poorly understood at the biochemical level. Structure data suggest that signals are transduced through intracellular loops of the TMDs that slot into grooves on the NBDs. At the base of these grooves is the Q loop. We therefore mutated the eponymous glutamine in one or both NBD Q loops and measured the effect on conformation and function by using a conformation-sensitive antibody (UIC2) and a fluorescent drug (Bodipy-verapamil), respectively. We showed that the double mutant is trapped in the inward-open state, which binds the drug, but cannot couple to the ATPase cycle. Our data also describe marked redundancy within the transport mechanism, because single-Q-loop mutants are functional for Bodipy-verapamil transport. This result allowed us to elucidate transduction pathways from twin drug-binding cavities to the Q loops using point mutations to favor one cavity over the other. Together, the data show that the Q loop is the central flexion point where the aspect of the drug-binding cavities is coupled to the ATP catalytic cycle.
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No. Sentence Comment
74 Plasmids Mutations were introduced into a plasmid encoding human ABCB1 with a C-terminal hexahistidine tag (pCIneo-wtABCB1-6His; ref. 25) by site-directed mutagenesis (QuikChange XL; Stratagene, La Jolla, CA, USA) using the following oligonucleotides: Q132A, 5=-GGTTGCTGCTTACATCGCGGTTTCATTTTGGTGC- 3=; Q132R, 5=-GGTTGCTGCTTACATTCGAGTTTCATTTTG- GTGC-3=; Q475A, 5=-GGGAAATCATTGGTGTGGTGAGTGCT- GAGCCTGTATTGTTTGCCACCACG-3=; Q773A, 5=-GGAATTA- TTTCTTTTATTACATTTTTCCTTGCGGGTTTCACATTTG- GCAAAGCTGG-3=; Q773R, 5=-GGAATTATTTCTTTTATTA- CATTTTTCCTTCGAGGTTTCACATTTGGCAAAGCTGG-3=; Q1118A, 5=-GGGCATCGTGTCCGCGGAACCCATCCTGTTTG-3=; E556Q, 5=-CCCCAAGATCCTCCTGCTTGATCAGGCCACGT- CAGCCTTGG-3=; and E1201Q, 5=-CAGCCTCATATTTTGCTTCT- TGATCAGGCCACGTCAGCTCTGGATAC-3=.
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ABCB1 p.Gln1118Ala 25016028:74:568
status: NEW121 The Q475A/ Q1118A mutant bound Bodipy-verapamil in the plasma membrane but did not efflux the drug; thus, it accumulated in the plasma membrane of cells expressing the double-Q-loop mutant ABCB1 and also in intracellular compartments (middle panels).
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ABCB1 p.Gln1118Ala 25016028:121:11
status: NEW132 RESULTS Q loops of the NBDs are essential for drug efflux, but redundancy is built into the molecular mechanism Site-directed mutagenesis was used to introduce glutamine-to-alanine mutations into NBD1 (NBD1-Q475A) and NBD2 (NBD2-Q1118A) and into both NBDs of human ABCB1.
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ABCB1 p.Gln1118Ala 25016028:132:229
status: NEW142 Cells expressing the single-NBD Q-loop mutants NBD1-Q475A or NBD2-Q1118A, accumulated only low levels of Bodipy-verapamil, similar, but not identical with, those expressing wild-type ABCB1 (Fig. 2C).
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ABCB1 p.Gln1118Ala 25016028:142:66
status: NEW144 In contrast, cells expressing the double mutant Q475A/Q1118A exhibited no efflux activity and, rather surprisingly and reproducibly, accumulated more Bodipy-verapamil than did the mock-transfected Figure 4.
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ABCB1 p.Gln1118Ala 25016028:144:54
status: NEW152 (The raw dot plot data also show that Bodipy-verapamil accumulation increased linearly with increasing expression of Q475A/ Q1118A; Supplemental Fig. S1.) Double-Q-loop Q475A/Q1118A mutant is trapped in the inward-open conformation The extracellular loops of human ABCB1 form a discontinuous epitope for the antibody UIC2, characterized by Igor Roninson and colleagues (38) and others (39).
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ABCB1 p.Gln1118Ala 25016028:152:124
status: NEWX
ABCB1 p.Gln1118Ala 25016028:152:175
status: NEW156 UIC2 reproducibly binds most readily to the Q475A/Q1118A mutant, suggesting that it adopts a conformation consistent with the inward-open state that would be expected to have a high affinity for UIC2.
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ABCB1 p.Gln1118Ala 25016028:156:50
status: NEW161 In contrast, cells expressing the Q475A/Q1118A mutant accumulated Bodipy-verapamil in both the intracellular compartments and the plasma membrane, where it colocalized with the 4E3 staining (Fig. 3, middle panels).
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ABCB1 p.Gln1118Ala 25016028:161:40
status: NEW163 The microscopy data are therefore consistent with the UIC2-binding data and suggest that the Q475A/ Q1118A mutant adopts the inward-open conformation, which, in the absence of the 2 Q-loop glutamines, cannot form the NBD-NBD interface and provides additional drug-binding sites in the plasma membrane, which explains the increased accumulation of Bodipy-verapamil in these cells.
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ABCB1 p.Gln1118Ala 25016028:163:100
status: NEW175 The drug-stimulated ATPase activity of the single-Q-loop mutants was reduced to 9.5 and 8.1% of the wild-type activity for the NBD1-Q475A and NBD2-Q1118A mutants, respectively, and the double mutant was inactive.
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ABCB1 p.Gln1118Ala 25016028:175:147
status: NEW177 In vitro ATPase data for NBD1-Q475A, NBD2-Q1118A, and wild-type ABCB1 Mutant af9;Nicardipine (50 òe;M) afa;Nicardipine Relative ATPase activity (%)a Km (mM) Vmax (nmol Pi/min/mg) Km (mM) Vmax (nmol Pi/min/mg) Wild type 0.746 afe; 0.22 1691 afe; 228 0.327 afe; 0.49 151 afe; 54 100 Q475A 2.37 afe; 1.64 162 afe; 70*** 0.284 afe; 0.19 8.0 afe; 1.7* 9.5 Q1118A 2.34 afe; 1.39 138 afe; 51*** 0.573 afe; 0.31 8.2 afe; 1.7* 8.1 Data are averages afe; se of c56;4 independent experiments.
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ABCB1 p.Gln1118Ala 25016028:177:42
status: NEWX
ABCB1 p.Gln1118Ala 25016028:177:385
status: NEW190 A) ECARs of HEK293T cells expressing wild-type (red), NBD1-Q475A (purple), or NBD2-Q1118A (orange) ABCB1 and mock-transfected cells (blue).
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ABCB1 p.Gln1118Ala 25016028:190:83
status: NEW193 Representative plots from cells expressing wild-type (B), NBD1-Q475A (C), NBD2-Q1118A (D), and Q475A/Q1118A (E) ABCB1.
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ABCB1 p.Gln1118Ala 25016028:193:79
status: NEWX
ABCB1 p.Gln1118Ala 25016028:193:101
status: NEW197 The V1 for cells expressing the single mutants NBD1-Q475A and NBD2-Q1118A was 1.19and 1.13-fold above basal rates, respectively, corresponding to 50 and 35% of the ATPase activity of wild-type ABCB1.
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ABCB1 p.Gln1118Ala 25016028:197:67
status: NEW198 No increase in ECAR in the presence of verapamil was detected in cells expressing the Q-loop double mutant Q475A/Q1118A.
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ABCB1 p.Gln1118Ala 25016028:198:113
status: NEW212 To test whether each verapamil-binding cavity was coupled to the NBDs via a specific Q loop, we combined the single drug cavity mutants TMD1-Q132R and TMD2- Q773R with the NBD Q-loop mutants NBD1-Q475A and NBD2-Q1118A and compared their transport activity (Fig. 6, striped bars) with that of the drug cavity mutants and also the single-and double-Q-loop mutants (Fig. 6, light gray bars; note that the double-Q-loop mutant has a fold difference that is b0d;1 because it provides additional binding sites for Bodipy-verapamil in the membrane).
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ABCB1 p.Gln1118Ala 25016028:212:211
status: NEW213 The drug cavity mutant TMD1-Q132R combined synergistically with NBD1-Q475A to significantly reduce Bodipy-verapamil export activity to 25% of wild-type activity, but in combination with NBD2-Q1118A, the transporter retained the full level of activity of each single mutant.
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ABCB1 p.Gln1118Ala 25016028:213:191
status: NEW214 (At 85% of the wild type, this activity was not significantly different from that of the Q132R mutant or the Q1118A mutant.)
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ABCB1 p.Gln1118Ala 25016028:214:109
status: NEW216 In contrast, TMD2- Q773R combined synergistically with both NBD1-Q475A and NBD2-Q1118A to reduce Bodipy-verapamil export activity to 22 and 34% of the wild-type activity, respectively, showing that the wild-type Q132-lined verapamil-binding cavity of these mutants is coupled to and requires the Q loops of both NBDs to trigger efflux.
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ABCB1 p.Gln1118Ala 25016028:216:80
status: NEW218 Cytosensor data for NBD1-Q475A, NBD2-Q1118A, Q475A/Q1118A, and wild-type ABCB1 Mutant K1 (òe;M) V1 (fold increase) V1 (% wild-type) K2 (òe;M) V2 (fold increase) Wild type 0.56 afe; 0.03 1.38 afe; 0.01 100 23.05 afe; 3.44 1.09 afe; 0.01 Q475A 0.91 afe; 0.10*** 1.19 afe; 0.02*** 50.2 26.54 afe; 4.84 0.94 afe; 0.02*** Q1118A 0.95 afe; 0.17** 1.13 afe; 0.02*** 35.6 84.81 afe; 30.36*** 0.88 afe; 0.06*** Q475A/Q1118A ND ND ND ND ND Data are averages afe; se of c56;3 independent experiments.
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ABCB1 p.Gln1118Ala 25016028:218:37
status: NEWX
ABCB1 p.Gln1118Ala 25016028:218:51
status: NEWX
ABCB1 p.Gln1118Ala 25016028:218:349
status: NEWX
ABCB1 p.Gln1118Ala 25016028:218:452
status: NEW253 ߤ Cells expressing the Q475A/Q1118A mutant ABCB1 accumulated more Bodipy-verapamil than did the nontransfected cells, giving a ratio of b0d;1. ns, not significant.
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ABCB1 p.Gln1118Ala 25016028:253:35
status: NEW