ABCMdb

ABCB1 p.Gln132Arg

Predicted by SNAP2:	A: D (85%), C: D (80%), D: D (91%), E: D (91%), F: D (91%), G: D (85%), H: D (85%), I: D (91%), K: D (95%), L: D (91%), M: D (85%), N: D (91%), P: D (95%), R: D (91%), S: D (80%), T: D (91%), V: D (91%), 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 dissection of dual pseudosymmetric solut... Mol Pharmacol. 2011 Mar;79(3):443-52. Epub 2010 Dec 21. Parveen Z, Stockner T, Bentele C, Pferschy S, Kraupp M, Freissmuth M, Ecker GF, Chiba P
Molecular dissection of dual pseudosymmetric solute translocation pathways in human P-glycoprotein.
Mol Pharmacol. 2011 Mar;79(3):443-52. Epub 2010 Dec 21., [PMID:21177413]

Abstract [show]
The human multispecific drug efflux transporter P-glycoprotein (P-gp) causes drug resistance and modulates the pharmacological profile of systemically administered medicines. It has arisen from a homodimeric ancestor by gene duplication. Crystal structures of mouse MDR1A indicate that P-gp shares the overall architecture with two homodimeric bacterial exporters, Sav1866 and MsbA, which have complete rotational symmetry. For ATP-binding cassette transporters, nucleotide binding occurs in two symmetric positions in the motor domains. Based on the homology with entirely symmetric half-transporters, the present study addressed the key question: can biochemical evidence for the existence of dual drug translocation pathways in the transmembrane domains of P-gp be found? P-gp was photolabeled with propafenone analogs, purified, and digested proteolytically, and peptide fragments were identified by high-resolution mass spectrometry. Labeling was assigned to two regions in the protein by projecting data into homology models. Subsequently, symmetric residue pairs in the putative translocation pathways were identified and replaced by site-directed mutagenesis. Transport assays corroborated the existence of two pseudosymmetric translocation pathways. Although rhodamine123 has a preference to take one path, verapamil, propafenones, and vinblastine preferentially use the other. Two major findings ensued from this study: the existence of two solute translocation pathways in P-gp as a reflection of evolutionary origin from a homodimeric ancestor and selective but not exclusive use of one of these pathways by different P-gp solutes. The pseudosymmetric behavior reconciles earlier kinetic and thermodynamic data, suggesting an alternative concept of drug transport by P-gp that will aid in understanding the off-target quantitative structure activity relationships of P-gp interacting drugs.

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Sentences [show]
No. Sentence Comment
52 Site-directed mutagenesis was performed at positions 132 and 773 of hexahistidine-tagged human P-gp cloned into the entry vector pENTR4-MDR1-His6 to generate the Q132A, Q132R, Q773A, Q773R, and the Q132A/Q773A mutants using the following forward and reverse primers: Q132A: forward, 5Ј-CTG CTT ACA TTG CGG TTT CAT TTT GG-3Ј; reverse, 5Ј-CCA AAA TGA AAC CGC AAT GTA AGC AG-3Ј; Q132R: forward, 5Ј-GCT GCT TAC ATT CGT GTT TCA TTT TG-3Ј; reverse, 5Ј-CAA AAT GAA ACA CGA ATG TAA GCA GC-3Ј; Q773A: forward, 5Ј-CAT TTT TCC TTG CGG GTT TCA CAT TTG GC-3Ј; reverse, 5Ј-GCC AAA TGT GAA ACC CGC AAG GAA AAA TG-3Ј; Q773R: forward, 5Ј-CAT TTT TCC TTC GAG GTT TCA CAT TTG-3Ј; reverse, 5Ј-CAT TTT TCC TTC GAG GTT TCA CAT TTG-3Ј. Login to comment
53 Double mutant Q132R/Q773R was generated by restriction digestion of pENTR4-Q132R and pENTR4-Q773R with SalI and EcoRI and subsequent ligation of the mutated fragment from pENTR4-Q773R into pENTR4-Q132R using T4 DNA ligase (rapid DNA ligation kit; Fermentas, Vienna, Austria). Login to comment
137 Rhodamine123 Efflux in Q132R/A and Q773R/A Mutants. Login to comment
138 In an initial series of experiments, we monitored rhodamine123 efflux in HEK-293/EBNA cells transiently transfected with either wild-type P-gp or the Q132R and Q773R mutants. Login to comment
161 Effect of Propafenone Analogs on Rhodamine123 Efflux in Q132R/A and Q773R/A Mutants. Login to comment
172 The IC50 value for wild-type P-gp was found to be 0.11 Ϯ 0.06 ␮M (black), whereas mutant Q132R showed an almost 7-fold higher IC50 value of 0.71 Ϯ 0.46 ␮M (green). Login to comment
177 IC50 values for wild type (black) and mutants Q132R (green) and Q773R (orange) (Fig. 5, Table 2) were indistinguishable, demonstrating that uncharged P-gp solutes were not affected by introduction of a positive charge in either position 132 or 773. Login to comment
181 Effect of Verapamil and Vinblastine on Rhodamine123 Efflux in Q132R/A and Q773R/A Mutants. Login to comment
182 In a third series of experiments, we evaluated the effect of verapamil and vinblastine on rhodamine123 transport in wild type and mutants Q132R and Q773R. Login to comment
200 In contrast, the IC50 values for the Q132R and Q773R mutants were 1.24 Ϯ 0.13 and 0.14 Ϯ 0.04 ␮M, respectively. Login to comment
202 The respective IC50 values were 2.68 Ϯ 0.93 ␮M for wild type, 0.45 Ϯ 0.16 ␮M for mutant Q773R, and 9.19 Ϯ 1.2 ␮M for the Q132R mutant. Login to comment
232 Q132R (green) and Q773R (orange) show transport rates of 52 Ϯ 13 and 24 Ϯ 6% of wild type, respectively (error propagation accounted for). Login to comment
233 The double mutant Q132R/Q773R (cyan) shows flux rates that are equal to simple diffusion (0.7-0.8/s). Login to comment
246 Both the Q132R and the Q773R mutant showed reduced transport activity for rhodamine123, which, however, was more pronounced in the Q773R mutant. Login to comment
265 Wild-type P-gp, black curve; Q132R, green curve; Q773R, orange curve. Login to comment
285 Wild-type P-gp, black curve; Q132R, green curve; Q773R, orange curve. Login to comment
289 Black curve, wild-type P-gp; green curve, Q132R; orange curve, Q773R. Login to comment
295 Black curve, wild-type P-gp; green curve, Q132R; orange curve, Q773R. Login to comment
299 Mutation IC50 Verapamil Vinblastine GPV31 GPV366 ␮M Wild-type P-gp 0.43 Ϯ 0.10 2.68 Ϯ 0.93 0.11 Ϯ 0.06 2.60 Ϯ 0.69 Q132A 0.30 Ϯ 0.07 2.58 Ϯ 0.31 0.10 Ϯ 0.03 2.57 Ϯ 0.69 Q773A 0.42 Ϯ 0.15 2.80 Ϯ 0.12 0.11 Ϯ 0.05 2.40 Ϯ 1.47 Q132A/Q773A 0.44 Ϯ 0.16 2.24 Ϯ 0.43 0.12 Ϯ 0.01 2.87 Ϯ 0.63 Q132R 1.24 Ϯ 0.13* 9.19 Ϯ 1.20* 0.71 Ϯ 0.46* 2.64 Ϯ 0.59 Q773R 0.14 Ϯ 0.04* 0.45 Ϯ 0.16* 0.02 Ϯ 0.01* 2.09 Ϯ 0.90 * P Ͻ 0.05, significantly different from wild-type values. Login to comment

[hide] Pore-exposed tyrosine residues of P-glycoprotein a... Mol Pharmacol. 2014 Mar;85(3):420-8. doi: 10.1124/mol.113.088526. Epub 2013 Dec 23. Donmez Cakil Y, Khunweeraphong N, Parveen Z, Schmid D, Artaker M, Ecker GF, Sitte HH, Pusch O, Stockner T, Chiba P
Pore-exposed tyrosine residues of P-glycoprotein are important hydrogen-bonding partners for drugs.
Mol Pharmacol. 2014 Mar;85(3):420-8. doi: 10.1124/mol.113.088526. Epub 2013 Dec 23., [PMID:24366667]

Abstract [show]
The multispecific efflux transporter, P-glycoprotein, plays an important role in drug disposition. Substrate translocation occurs along the interface of its transmembrane domains. The rotational C2 symmetry of ATP-binding cassette transporters implies the existence of two symmetry-related sets of substrate-interacting amino acids. These sets are identical in homodimeric transporters, and remain evolutionary related in full transporters, such as P-glycoprotein, in which substrates bind preferentially, but nonexclusively, to one of two binding sites. We explored the role of pore-exposed tyrosines for hydrogen-bonding interactions with propafenone type ligands in their preferred binding site 2. Tyrosine 953 is shown to form hydrogen bonds not only with propafenone analogs, but also with the preferred site 1 substrate rhodamine123. Furthermore, an accessory role of tyrosine 950 for binding of selected propafenone analogs is demonstrated. The present study demonstrates the importance of domain interface tyrosine residues for interaction of small molecules with P-glycoprotein.

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Sentences [show]
No. Sentence Comment
54 Furthermore, Q132R and Q773R mutations were introduced to the constructs above by using the following: Q132R- forward, 59-GCTGCTTACATTCGTGTTTCATTTTG-39; Q132R-reverse, 59- CAAAATGAAACACGAATGTAAGCAGC-39; Q773R-forward, 59-CAT TTTTCCTTCGAGGTTTCACATTTG-39; and Q773R-reverse, 59-CA TTTTTCCTTCGAGGTTTCACATTTG-39. Login to comment
114 All mutants were detected at the plasma membrane, although the expression levels of Q132R/Y950F, Q773R/Y950F, and Y307F/Y310F were reduced (Supplemental Fig. 5). Login to comment
120 Access of rh123 to binding sites 1 and 2 can be controlled by the introduction of positive charges (arginines) in the access path to the ligand binding sites. We previously showed that the Q132R mutation blocks access to site 2 for rh123, whereas the Q773R mutation prevents rh123 access to site 1 (Parveen et al., 2011). Login to comment
125 This is illustrated by comparable transport activity of the Q132R/Y950F, Q132R/Y953F, and Q132R/ Y950F/Y953F mutants. Login to comment
155 The at least 3-fold difference in IC50 values for wild-type protein (GPV005, 518 6 141 nM; and GPV031, 85 6 22 nM) (Fig. 3, B and C) and the Q132R mutant (GPV005, 1414 6 215 nM; and GPV031, 521 6 183 nM) (Fig. 4, A and B) demonstrates that an arginine residue in position 132 prevents access of protonatable compounds to site 2. Login to comment
163 show that for the Q132R/Y950F mutant, IC50 values were similar to those observed in the Q132R background alone. Login to comment
164 However, IC50 values were still higher in the Q132R/Y953F mutation. Login to comment
168 For the nonprotonatable acid amide GPV366, a higher IC50 value was observed in the Q132R/Y953F mutant (Q132R, 1074 6 282 nM; and Q132R/Y953F, 3261 6 965 nM), whereas the Y950F mutation in the same background did not affect potency (966 6 259 nM) (Fig. 4C). Login to comment
169 Although the pattern was similar to that observed in the wild-type background, the fold change was lower in the Q132R background and the IC50 value was somewhat lower in the protein containing the Q132R mutation than for wild-type. Login to comment
170 A possible explanation for this effect is the lack of competition of GPV366 in the preferred binding site 2 with rh123, because rh123 binding to site 2 is abolished by the Q132R mutation. Login to comment
171 The higher IC50 values in the Q132R/Y950F/Y953F mutant were not due to an additive effect of the two tyrosine mutations on binding of propafenones. Login to comment
173 Certainly, the effect is not brought about by a global perturbance of protein structure and function, because the Q132R mutant and the Q132R/Y950F/Y953F triple mutant showed comparable rh123 transport rates (Fig. 2). Login to comment
189 The tyrosine mutants were generated in the Q132R mutation background. Login to comment
191 *P , 0.05; **P , 0.01; ***P , 0.001, compared with Q132R mutation. Login to comment
193 Identical transport rates were found for the Q132R and the Y953F mutants in the Q132R background. Login to comment
200 IC50 values were therefore expected to be the same in single and double tyrosine mutants generated in the Q132R background. Login to comment
201 Indeed, no difference was observed between the Q132R single mutant and the Q132R/Y950F double mutant. Login to comment
205 The uncharged ligand species can still have access to site 2, which has been deselected for the protonated ligand species by introducing the Q132R mutation. Login to comment
212 For GPV005, mutant Q132R/Y953F showed a 1.5-fold higher IC50 value than the Q132R mutation, whereas the fold changes were 2.1-fold for GPV031 and 3.0-fold for uncharged GPV366. Login to comment
214 However, such an effect was not observed in the Q132R background (Fig. 4, A and B). Login to comment
217 The triple Q132R/Y950F/Y953F mutant showed a further increase in IC50 values for all compounds, which is obviously not due to interaction with tyrosines. Login to comment

[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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Sentences [show]
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=. Login to comment
207 The single mutant TMD1-Q132R effluxed the Bodipy-verapamil with 70% efficiency of the wild-type protein, whereas the TMD2- Q773R was fully active and indistinguishable from wild-type ABCB1 (Fig. 6, dark gray bars, in which transport data are presented as the fold reduction in Bodipy-verapamil accumulation compared to mock-transfected cells). Login to comment
209 The double-arginine mutant Q132R/ Q773R retained the ability to export Bodipy-verapamil, but it was significantly reduced to 42% (Pb0d;0.001) of wild-type ABCB1 activity. Login to comment
210 This residual activity is most likely due to the partial masking of the positive charge on verapamil by the Bodipy moiety, such that electrostatic repulsion from Q132R or Q773R is incomplete. Login to comment
212 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). Login to comment
213 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. Login to comment
214 (At 85% of the wild type, this activity was not significantly different from that of the Q132R mutant or the Q1118A mutant.) Login to comment
215 This observation shows that the wild-type Q773- lined verapamil-binding cavity of the Q132R mutant is dedicated to, and only requires, the NBD1 Q loop to couple to the ATP catalytic cycle. Login to comment
224 The single-Q-loop mutants, combined with the TMD mutants Q132R in TMD1 or Q773R in TMD2 that line the 2 verapamil-binding cavities (12), showed that Bodipy-verapamil bound to the cavity lined by TMD2-Q773 triggers conformation change to the inward-closed state via the conduit of the Q loop in NBD1. Login to comment