ABCB1 p.Gly984Cys
| Predicted by SNAP2: | A: D (63%), C: D (66%), D: D (85%), E: D (91%), F: D (91%), H: D (91%), I: D (91%), K: D (91%), L: D (91%), M: D (85%), N: D (85%), P: D (91%), Q: D (91%), R: D (91%), S: N (53%), T: D (75%), V: D (85%), W: D (91%), Y: D (91%), |
| Predicted by PROVEAN: | A: D, C: D, D: D, E: D, F: 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] Transmembrane segment 1 of human P-glycoprotein co... Biochem J. 2006 Jun 15;396(3):537-45. Loo TW, Bartlett MC, Clarke DM
Transmembrane segment 1 of human P-glycoprotein contributes to the drug-binding pocket.
Biochem J. 2006 Jun 15;396(3):537-45., 2006-06-15 [PMID:16492138]
Abstract [show]
P-glycoprotein (P-gp; ABCB1) actively transports a broad range of structurally unrelated compounds out of the cell. An important step in the transport cycle is coupling of drug binding with ATP hydrolysis. Drug substrates such as verapamil bind in a common drug-binding pocket at the interface between the TM (transmembrane) domains of P-gp and stimulate ATPase activity. In the present study, we used cysteine-scanning mutagenesis and reaction with an MTS (methanethiosulphonate) thiol-reactive analogue of verapamil (MTS-verapamil) to test whether the first TM segment [TM1 (TM segment 1)] forms part of the drug-binding pocket. One mutant, L65C, showed elevated ATPase activity (10.7-fold higher than an untreated control) after removal of unchanged MTS-verapamil. The elevated ATPase activity was due to covalent attachment of MTS-verapamil to Cys65 because treatment with dithiothreitol returned the ATPase activity to basal levels. Verapamil covalently attached to Cys65 appears to occupy the drug-binding pocket because verapamil protected mutant L65C from modification by MTS-verapamil. The ATPase activity of the MTS-verapamil-modified mutant L65C could not be further stimulated with verapamil, calcein acetoxymethyl ester or demecolcine. The ATPase activity could be inhibited by cyclosporin A but not by trans-(E)-flupentixol. These results suggest that TM1 contributes to the drug-binding pocket.
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No. Sentence Comment
41 A series of double cysteine mutants containing L65C in TM1 with another cysteine in TMD2 (C-terminal TMD containing TM7-TM12) predicted to line the drug-binding pocket [34] (i.e. F942C or T945C in TM11 and L975C, V981C, V982C, G984C or A985C in TM12) were also constructed for cross-linking analysis.
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ABCB1 p.Gly984Cys 16492138:41:227
status: NEW60 Disulphide cross-linking analysis Mutants L65C, F942C, T945C, L975C, V981C, V982C, G984C, A985C, L65C/F942C, L65C/T945C, L65C/975C, L65C/V981C, L65C/V982C, L65C/G984C and L65C/A985C were transiently expressed in HEK-293 cells.
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ABCB1 p.Gly984Cys 16492138:60:83
status: NEWX
ABCB1 p.Gly984Cys 16492138:60:161
status: NEW160 Accordingly, Figure 6 Disulphide cross-linking of P-gp mutants (A) Membranes were prepared from HEK-293 cells (A) expressing mutants L65C, L65C/T945C, L65C/V982C, L65C/G984C or L65C/A985C.
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ABCB1 p.Gly984Cys 16492138:160:170
status: NEW[hide] Transmembrane helix 12 modulates progression of th... Biochemistry. 2009 Jul 7;48(26):6249-58. Crowley E, O'Mara ML, Reynolds C, Tieleman DP, Storm J, Kerr ID, Callaghan R
Transmembrane helix 12 modulates progression of the ATP catalytic cycle in ABCB1.
Biochemistry. 2009 Jul 7;48(26):6249-58., 2009-07-07 [PMID:19456124]
Abstract [show]
Multidrug efflux pumps, such as P-glycoprotein (ABCB1), present major barriers to the success of chemotherapy in a number of clinical settings. Molecular details of the multidrug efflux process by ABCB1 remain elusive, in particular, the interdomain communication associated with bioenergetic coupling. The present investigation has focused on the role of transmembrane helix 12 (TM12) in the multidrug efflux process of ABCB1. Cysteine residues were introduced at various positions within TM12, and their effect on ATPase activity, nucleotide binding, and drug interaction were assessed. Mutation of several residues within TM12 perturbed the maximal ATPase activity of ABCB1, and the underlying cause was a reduction in basal (i.e., drug-free) hydrolysis of the nucleotide. Two of the mutations (L976C and F978C) were found to reduce the binding of [gamma-(32)P]-azido-ATP to ABCB1. In contrast, the A980C mutation within TM12 enhanced the rate of ATP hydrolysis; once again, this was due to modified basal activity. Several residues also caused reductions in the potency of stimulation of ATP hydrolysis by nicardipine and vinblastine, although the effects were independent of changes in drug binding per se. Overall, the results indicate that TM12 plays a key role in the progression of the ATP hydrolytic cycle in ABCB1, even in the absence of the transported substrate.
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No. Sentence Comment
67 This necessitated the centrifugation (100000g for 30 min) of 500 μL Table 1: Mutagenic Oligonucleotide Primers Used To Generate TM12 Mutationsa mutation primer sequence (50 -30 ) diagnostic restriction digest L976C GAGGATGTTCTAtgtGTATTTTCAGCTGTTG -SpeI F978C GTTCTACTAGTATgTTCtGCaGTTGTCTTTGGTG +PstI A980C CTACTAGTATTTTCAtgcGTTGTCTTTGGTGCCATGGCC -PvuII V982C CTAGTATTTTCAGCgGTTtgCTTTGGTGCCATGGCC -PvuII G984C GCTGTTGTCTTTtGTGCtATGGCCGTGG -NcoI M986C GTATTTGGTGCttgtGCtGTGGGGCAAGTC -NcoI V988C GGTGCCATGGCCtgtGGGCAAGTCAGTTC -BstXI G989C CTTTGGTGCCATGGCCGTGtGcCAAGTCAGTTCATTTGC +BstXI Q990C GGCCGTGGGGtgtGTCtcTTCATTTGCTCC +EarI a Primer sequences contain an introduced cysteine residue (bold) and additional silent mutations (lower case), with respect to the coding sequence that generates or removes the indicated restriction site.
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ABCB1 p.Gly984Cys 19456124:67:409
status: NEW151 There was, however, a drug-dependent difference on the stimulation of ATPase activity observed with the G984C and FIGURE 4: Maximal ATPase activity of TM12 mutant isoforms.
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ABCB1 p.Gly984Cys 19456124:151:104
status: NEW155 Table 2: Potency and Degree of Drug Stimulation of ATP Hydrolysis by ABCB1a nicardipine vinblastine EC50 (μM) fold stimulation EC50 (μM) fold stimulation Cys-less 4.1 ( 1.1 4.0 ( 0.6 5.91 ( 2.9 2.2 ( 0.2 L976C 5.2 ( 0.2 7.4 ( 1.4 10.0 ( 0.0 3.5 ( 0.6 F978C 24.1 ( 2.3b 9.5 ( 1.4 42.9 ( 4.3b 2.3 ( 0.5 A980C 3.4 ( 0.3 5.1 ( 0.9 12.3 ( 1.8 3.2 ( 0.8 V982C 5.8 ( 0.9 4.2 ( 0.5 2.0 ( 0.7 1.8 ( 0.2 G984C 37.6 ( 11.2b 16.2 ( 6.6b 6.7 ( 1.7 6.2 ( 2.3 M986C 9.2 ( 0.8 4.7 ( 1.1 15.0 ( 2.0b 2.8 ( 0.7 V988C 3.9 ( 0.6 3.1 ( 0.1 7.3 ( 2.3 1.9 ( 0.2 G989C 13.6 ( 1.5 5.1 ( 1.6 4.9 ( 0.9 2.4 ( 0.3 Q990C 6.9 ( 1.1 3.7 ( 1.0 NDc NDc S992C 4.9 ( 0.5 4.2 ( 0.6 7.1 ( 2.6 2.3 ( 0.4 F994C 1.7 ( 0.4 3.2 ( 0.8 5.9 ( 2.5 1.6 ( 0.3 a ATPase activity was plotted as a function of the drug concentration and potency (EC50) and degree of stimulation obtained by nonlinear regression of the dose-response relationship equation.
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ABCB1 p.Gly984Cys 19456124:155:406
status: NEW160 For G984C, nicardipine was associated with a reduction in potency and degree of stimulation, whereas the influence of vinblastine was similar to that in cysteine-less ABCB1.
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ABCB1 p.Gly984Cys 19456124:160:4
status: NEW[hide] Transmembrane helix 12 plays a pivotal role in cou... FEBS J. 2010 Oct;277(19):3974-85. doi: 10.1111/j.1742-4658.2010.07789.x. Epub 2010 Aug 20. Crowley E, O'Mara ML, Kerr ID, Callaghan R
Transmembrane helix 12 plays a pivotal role in coupling energy provision and drug binding in ABCB1.
FEBS J. 2010 Oct;277(19):3974-85. doi: 10.1111/j.1742-4658.2010.07789.x. Epub 2010 Aug 20., [PMID:20731718]
Abstract [show]
Describing the molecular details of the multidrug efflux process of ABCB1, in particular the interdomain communication associated with bioenergetic coupling, continues to prove difficult. A number of investigations to date have implicated transmembrane helix 12 (TM12) in mediating communication between the transmembrane domains and nucleotide-binding domains (NBDs) of ABCB1. The present investigation further addressed the role of TM12 in ABCB1 by characterizing its topography during the multidrug efflux process with the use of cysteine-directed mutagenesis. Cysteines were introduced at various positions along TM12 and assessed for their ability to covalently bind thiol-reactive fluorescent probes with differing physiochemical properties. By analysing each isoform in the basal, ATP-bound and posthydrolytic states, it was possible to determine how the local environment of TM12 alters during the catalytic cycle. Labelling with hydrophobic CM and zwitterionic BM was extensive throughout the helix in the basal, prehydrolytic and posthydrolytic states, suggesting that TM12 is in a predominantly hydrophobic environment. Overall, the carboxy region (intracellular half) of TM12 appeared to be more responsive to changes in the catalytic state of the protein than the amino region (extracellular half). Thus, the carboxy region of TM12 is suggested to be responsive to nucleotide binding and hydrolysis at the NBDs and therefore directly involved in interdomain communication. This data can be reconciled with an atomic-scale model of human ABCB1. Taken together, these results indicate that TM12 plays a key role in the progression of the ATP hydrolytic cycle in ABCB1 and, in particular, in coordinating conformational changes between the NBDs and transmembrane domains.
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No. Sentence Comment
82 The rate of labelling (i.e. t1 / 2) was divided into fast (L986C- G992C, average t1 / 2 $ 8 min) and slow (L976C- G984C, average t1 / 2 $ 25 min) kinetics between the carboxy-half and the amino-half, respectively.
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ABCB1 p.Gly984Cys 20731718:82:114
status: NEW92 However, two residues (G984C and M986C) in the central region of TM12 did display labelling above background, albeit with Lext values of approximately 20%.
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ABCB1 p.Gly984Cys 20731718:92:23
status: NEW139 Mutant CM BM FM Lext (%) t1 / 2 (min) Lext (%) t1 / 2 (min) Lext (%) t1 / 2 (min) L976C 38 ± 5 29 ± 12 66 ± 14 29 ± 18 - - A980C 53 ± 6 34 ± 1 54 ± 8 20 ± 9 - - V982C 98 ± 14 15 ± 6 164 ± 50 27 ± 17 - - G984C 73 ± 14 29 ± 6 84 ± 24 22 ± 7 13 ± 10 ND M986C 89 ± 30 25 ± 10 51 ± 5 3 ± 2 21 ± 2 ND V988C 53 ± 6 37 ± 18 221 ± 63 18 ± 12 - - G989C 64 ± 7 15 ± 6 21 ± 3 9 ± 2 - - S992C 55 ± 4 22 ± 6 51 ± 5 4 ± 1 32 ± 3 25 ± 5 F994C 51 ± 10 11 ± 9 111 ± 35 13 ± 10 129 ± 24 8 ± 3 Conformational changes - central region Two of the residues examined in the central region (G984C and M986C) of TM12 have been shown to accommodate partial labelling with FM, suggestive of aqueous accessibility in the basal state. At M986C, the extent of labelling with the hydrophilic probe was increased following the addition of nonhydrolysable nucleotide.
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ABCB1 p.Gly984Cys 20731718:139:263
status: NEWX
ABCB1 p.Gly984Cys 20731718:139:777
status: NEW143 G984C underwent a broadly similar shift in topography as M986C, although the degree of alteration was somewhat less striking.
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ABCB1 p.Gly984Cys 20731718:143:0
status: NEW164 ABCB1 isoform Catalytic intermediate CM BM FM L976C Basal ++ +++ ) AMP-PNP +++ ++ ) Vi trapped +++ +++ ) A980C Basal ++ ++ ) AMP-PNP +++ + ) Vi trapped +++ +++ ) V982C Basal +++ +++ ) AMP-PNP +++ +++ ) Vi trapped +++ +++ ) G984C Basal +++ +++ + AMP-PNP +++ +++ + Vi trapped +++ ++ ) M986C Basal +++ ++ + AMP-PNP ++ +++ ++ Vi trapped +++ ++ ) V988C Basal ++ +++ ) AMP-PNP +++ +++ ) Vi trapped +++ +++ ) G989C Basal ++ + ) AMP-PNP ++ ++ ) Vi trapped ++ + ) S992C Basal ++ ++ + AMP-PNP +++ +++ ++ Vi trapped ++ ++ + F994C Basal ++ +++ +++ AMP-PNP ++ +++ ++ Vi trapped +++ +++ + reflect localization at the membrane-solute interface.
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ABCB1 p.Gly984Cys 20731718:164:223
status: NEW183 The homology models predict that both V982C and G984C, located within the centre of the helix, experience little change in molecular environment upon ATP binding, which is in agreement with the biochemical data.
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ABCB1 p.Gly984Cys 20731718:183:48
status: NEW196 Surprisingly, although FM labels G984C, the homology model suggests that this residue faces into the lipid bilayer.
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ABCB1 p.Gly984Cys 20731718:196:33
status: NEW197 However, G984C is not in a very densely packed region, and it may be possible for FM to gain access to the residue via the translocation pore.
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ABCB1 p.Gly984Cys 20731718:197:9
status: NEW198 In addition, the loss of labelling of G984C with FM following progression to a vanadate-trapped state suggests that labelling is not optimal and therefore is very sensitive to even minor environmental changes.
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ABCB1 p.Gly984Cys 20731718:198:38
status: NEW228 The structures are shown in the panel as viewed from the translocation pore; the relative environments of V982C (cyan) and G984C (blue) are unaltered by nucleotide binding.
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ABCB1 p.Gly984Cys 20731718:228:123
status: NEW[hide] Identification of residues in the drug-binding sit... J Biol Chem. 1997 Dec 19;272(51):31945-8. Loo TW, Clarke DM
Identification of residues in the drug-binding site of human P-glycoprotein using a thiol-reactive substrate.
J Biol Chem. 1997 Dec 19;272(51):31945-8., 1997-12-19 [PMID:9405384]
Abstract [show]
We identified a thiol-reactive compound, dibromobimane (dBBn), that was a potent stimulator (8.2-fold) of the ATPase activity of Cys-less P-glycoprotein. We then used this compound together with cysteine-scanning mutagenesis to identify residues in transmembrane segment (TM) 6 and TM12 that are important for function. TM6 and TM12 lie close to each other in the tertiary structure and are postulated to be important for drug-protein interactions. The majority of P-glycoprotein mutants containing a single cysteine residue retained substantial amounts of drug-stimulated ATPase activity and were not inhibited by dBBn. The ATPase activities of mutants L339C, A342C, L975C, V982C, and A985C, however, were markedly inhibited (>60%) by dBBn. The drug substrates verapamil, vinblastine, and colchicine protected these mutants against inhibition by dBBn, suggesting that these residues are important for interaction of substrates with P-glycoprotein. We previously showed that residues Leu339, Ala342, Leu975, Val982, and Ala985 lie along the point of contact between helices TM6 and TM12, when both are aligned in a left-handed coiled coil (Loo, T. W., and Clarke, D. M. (1997) J. Biol. Chem. 272, 20986-20989). Taken together, these results suggest that the interface between TM6 and TM12 likely forms part of the potential drug-binding pocket in P-glycoprotein.
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No. Sentence Comment
83 There was no detectable activity with mutants S344C, G341C, and G984C, whereas mutants A342C, G346C, Q347C, A985C, G989C, and Q990C had much reduced activity (10-40%).
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ABCB1 p.Gly984Cys 9405384:83:64
status: NEW87 Mutants G341C and G984C, however, appeared to be degraded quite rapidly.
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ABCB1 p.Gly984Cys 9405384:87:18
status: NEW96 Mutants G341C, S344C, G346C, G984C, and G989C were not assayed because of their low or defective expression (Fig. 2B).
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ABCB1 p.Gly984Cys 9405384:96:29
status: NEW[hide] New light on multidrug binding by an ATP-binding-c... Trends Pharmacol Sci. 2006 Apr;27(4):195-203. Epub 2006 Mar 20. Shilling RA, Venter H, Velamakanni S, Bapna A, Woebking B, Shahi S, van Veen HW
New light on multidrug binding by an ATP-binding-cassette transporter.
Trends Pharmacol Sci. 2006 Apr;27(4):195-203. Epub 2006 Mar 20., [PMID:16545467]
Abstract [show]
ATP-binding-cassette (ABC) multidrug transporters confer multidrug resistance to pathogenic microorganisms and human tumour cells by mediating the extrusion of structurally unrelated chemotherapeutic drugs from the cell. The molecular basis by which ABC multidrug transporters bind and transport drugs is far from clear. Genetic analyses during the past 14 years reveal that the replacement of many individual amino acids in mammalian multidrug resistance P-glycoproteins can affect cellular resistance to drugs, but these studies have failed to identify specific regions in the primary amino acid sequence that are part of a defined drug-binding pocket. The recent publication of an X-ray crystallographic structure of the bacterial P-glycoprotein homologue MsbA and an MsbA-based homology model of human P-glycoprotein creates an opportunity to compare the original mutagenesis data with the three-dimensional structures of transporters. Our comparisons reveal that mutations that alter specificity are present in three-dimensional 'hotspot' regions in the membrane domains of P-glycoprotein.
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No. Sentence Comment
78 Single-cysteine mutants in human P-glycoprotein that are protected from cross-linking to cysteine-reactive MTS substrate analogues by the non-reactive substratea P-glycoprotein residueb Corresponding residue in V. cholera MsbA Cysteine-reactive substrate I340C (6) G293 MTS-rhodamine A841C (9) A151 MTS-rhodamine L975C (12) T285 MTS-rhodamine V981C (12) M291 MTS-rhodamine V982C (12) F292 MTS-rhodamine S222C (4) A175 MTS-verapamil L339C (6) M291 MTS-verapamil A342C (6) M295 MTS-verapamil I868C (10) F180 MTS-verapamil F942C (11) Q256 MTS-verapamil T945C (11) A259 MTS-verapamil G984C (12) L294 MTS-verapamil a Data adapted from [24,2].
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ABCB1 p.Gly984Cys 16545467:78:580
status: NEW76 Single-cysteine mutants in human P-glycoprotein that are protected from cross-linking to cysteine-reactive MTS substrate analogues by the non-reactive substratea P-glycoprotein residueb Corresponding residue in V. cholera MsbA Cysteine-reactive substrate I340C (6) G293 MTS-rhodamine A841C (9) A151 MTS-rhodamine L975C (12) T285 MTS-rhodamine V981C (12) M291 MTS-rhodamine V982C (12) F292 MTS-rhodamine S222C (4) A175 MTS-verapamil L339C (6) M291 MTS-verapamil A342C (6) M295 MTS-verapamil I868C (10) F180 MTS-verapamil F942C (11) Q256 MTS-verapamil T945C (11) A259 MTS-verapamil G984C (12) L294 MTS-verapamil a Data adapted from [24,25].
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ABCB1 p.Gly984Cys 16545467:76:580
status: NEW