ABCB1 p.Gly324Cys
| Predicted by SNAP2: | A: N (66%), C: D (66%), D: N (82%), E: N (93%), F: D (75%), H: N (61%), I: D (53%), K: N (87%), L: D (59%), M: N (53%), N: N (93%), P: N (53%), Q: N (82%), R: N (61%), S: N (82%), T: N (78%), V: N (53%), W: D (85%), Y: D (75%), |
| Predicted by PROVEAN: | A: N, C: D, D: N, E: N, F: N, H: N, I: D, K: N, L: D, M: N, N: N, P: N, Q: N, R: N, S: N, T: N, V: N, W: D, Y: N, |
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[hide] Cysteine-scanning mutagenesis provides no evidence... EMBO J. 1999 Dec 1;18(23):6800-8. Blott EJ, Higgins CF, Linton KJ
Cysteine-scanning mutagenesis provides no evidence for the extracellular accessibility of the nucleotide-binding domains of the multidrug resistance transporter P-glycoprotein.
EMBO J. 1999 Dec 1;18(23):6800-8., 1999-12-01 [PMID:10581253]
Abstract [show]
Multidrug resistance of cancer cells is, at least in part, conferred by overexpression of P-glycoprotein (P-gp), a member of the ATP-binding cassette (ABC) superfamily of active transporters. P-gp actively extrudes chemotherapeutic drugs from cells, thus reducing their efficacy. As a typical ABC transporter, P-gp has four domains: two transmembrane domains, which form a pathway through the membrane through which substrates are transported, and two hydrophilic nucleotide-binding domains (NBDs), located on the cytoplasmic side of the membrane, which couple the energy of ATP hydrolysis to substrate translocation. It has been proposed that the NBDs of ABC transporters, including the histidine permease of Salmonella typhimurium and the cystic fibrosis transmembrane conductance regulator, are accessible from the extracellular surface of the cell, spanning the membrane directly or potentially contributing to the transmembrane pore. Such organization would have significant implications for the transport mechanism. We determined to establish whether the NBDs of P-gp are exposed extracellularly and which amino acids are accessible, using cysteine-scanning mutagenesis and limited proteolysis. In contrast to other transporters, the data provided no evidence that the P-gp NBDs are exposed to the cell surface. The implications for the structure and mechanism of P-gp and other ABC transporters are discussed.
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No. Sentence Comment
36 Two additional single-cysteine (SC) mutants (N280C and G324C), known to be located in an intracellular and extracellular loop of TMD1, respectively (Loo and Clarke, 1995), were also generated as controls.
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ABCB1 p.Gly324Cys 10581253:36:55
status: NEW38 P-gp-cys- and 21 of the 30 SC mutants (including the control mutants SC-N280C and SC-G324C) expressed full-length protein in both the lower molecular weight non-glycosylated and the higher molecular weight glycosylated forms (Figure 2).
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ABCB1 p.Gly324Cys 10581253:38:85
status: NEW66 To establish the validity of the assay, SC-P-gp mutants G324C (known to be exposed in an extracellular loop of TMD1) and N280C (known to be located in an intracellular loop of TMD1) were used as positive and negative controls, respectively.
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ABCB1 p.Gly324Cys 10581253:66:56
status: NEW67 In intact cells, the higher molecular weight, glycosylated P-gp-G324C was biotinylated while neither form of P-gp- N280C was biotinylated (Figure 4A), as expected.
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ABCB1 p.Gly324Cys 10581253:67:64
status: NEW69 In this case, both the mature and immature (nonglycosylated) forms of G324C and N280C P-gp were labelled equivalently (Figure 4B): immature P-gp is likely to be confined to intracellular membranes and so accessible to BM only after permeabilization of the cell.
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ABCB1 p.Gly324Cys 10581253:69:70
status: NEW190 Oligonucleotides used for site-directed mutagenesis pSC-name Diagnostic restriction site Mutagenic oligonucleotide sequence 5Ј-3Ј N280C ϩAvaI GGTACAACAAATGTCTCGAGGAAGCTAAAAG G324C -AflIII CCTTGGTCTTATCATGTGAATATTCTATTGG S403C ϩPstI CTTCAGTTACCCCTGCAGAAAAGAAGTTAAG S419C ϩEco57I GAACCTGAAAGTGCAGTGTGGGCAGACG Q456C ϩBstEI GTTGATGGATGCGATATCCGGACCATAAATG F465C ϩBanI ATGTAAGGTGCCTACGGGAA I469C ϩNsiI CTACGGGAATGCATTGGTGT S474C ϩHpaII GGTGTCGTGTGTCAGGAACCGGTATTGTTT T482C ϩBsrGI GTATTGTTTGCCTGTACAATAGCTGAAAAC I488C ϩEclXI GCTGAAAACTGTCGCTACGGCCGTGAAAATG Y490C none ACATTCGCTGTGGCCGTGA V495C ϩBsrGI GGCCGTGAAAATTGTACAATGGATGAGATTG D498C -NcoI GTCACCATGTGTGAGATTGAG I500C ϩBsmI GTCACCATGGATGAATGCGAGAAAGCTGTC K502C ϩBsmI GGATGAGATTGAATGCGCTGTCAAGGAAG N508C ϩFspI GTCAAGGAAGCATGCGCATATGACTTTATC D511C ϩAflIII GGAAGCCAACGCGTATTGCTTTATCATG K515C ϩAflIII GAAGCCAACGCGTATGACTTTATCATGTGCCTGCCTCAT K519C ϩStyI GAAACTGCCTCATTGCTTTGACACCTTGGTTGGAGAGAGAG A529C ϩBanI GAGAGAGGGTGCCAGTTGAG K536C ϩBlpI GAGAGAGAGGCGCCCAGTTGAGTGGTGGGCAGTGCCAGAGGATCG R547C ϩBssSI GCACGTGCCCTCGTGTGCAACCCCAAG P549C ϩSspI TGGTTCGCAACTGCAAAATATTCCTGCTGGA L554C ϩSspI CGCAACCCCAAAATATTGCTGTGCGATGAGGCCACG S565C ϩBsmI GACACAGAATGCGAAGCAG V569C ϩBsmI GACACAGAATGCGAAGCAGTGTGTCAGGTGG K578C ϩBsiEI GATAAGGCCAGATGCGGCCGGACCACC R580C -MslI GCCACAAAAGGTTGCACGACCATTGTGATA T581C ϩApaLI GAAAAGGTCGGTGCACCATTGTG E638C ϩEclXI GAAGTTGAATTATGCAATGCGGCCGATGAATC 'ϩ` represents the introduction of a new restriction endonuclease site; '-` represents the removal of an existing site.
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ABCB1 p.Gly324Cys 10581253:190:192
status: NEW[hide] Repacking of the transmembrane domains of P-glycop... EMBO J. 2001 Oct 15;20(20):5615-25. Rosenberg MF, Velarde G, Ford RC, Martin C, Berridge G, Kerr ID, Callaghan R, Schmidlin A, Wooding C, Linton KJ, Higgins CF
Repacking of the transmembrane domains of P-glycoprotein during the transport ATPase cycle.
EMBO J. 2001 Oct 15;20(20):5615-25., 2001-10-15 [PMID:11598005]
Abstract [show]
P-glycoprotein (P-gp) is an ABC (ATP-binding cassette) transporter, which hydrolyses ATP and extrudes cytotoxic drugs from mammalian cells. P-gp consists of two transmembrane domains (TMDs) that span the membrane multiple times, and two cytoplasmic nucleotide-binding domains (NBDs). We have determined projection structures of P-gp trapped at different steps of the transport cycle and correlated these structures with function. In the absence of nucleotide, an approximately 10 A resolution structure was determined by electron cryo-microscopy of two-dimensional crystals. The TMDs form a chamber within the membrane that appears to be open to the extracellular milieu, and may also be accessible from the lipid phase at the interfaces between the two TMDs. Nucleotide binding causes a repacking of the TMDs and reduction in drug binding affinity. Thus, ATP binding, not hydrolysis, drives the major conformational change associated with solute translocation. A third distinct conformation of the protein was observed in the post-hydrolytic transition state prior to release of ADP/P(i). Biochemical data suggest that these rearrangements may involve rotation of transmembrane alpha-helices. A mechanism for transport is suggested.
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No. Sentence Comment
138 As expected, the drug-stimulated ATPase activities of cysteine-less P-gp and the control (G324C, which has a single cysteine in an extracellular loop; Blott et al., 1999) were not signi®cantly affected by either reagent.
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ABCB1 p.Gly324Cys 11598005:138:90
status: NEW[hide] The topography of transmembrane segment six is alt... J Biol Chem. 2004 Aug 13;279(33):34913-21. Epub 2004 Jun 10. Rothnie A, Storm J, Campbell J, Linton KJ, Kerr ID, Callaghan R
The topography of transmembrane segment six is altered during the catalytic cycle of P-glycoprotein.
J Biol Chem. 2004 Aug 13;279(33):34913-21. Epub 2004 Jun 10., 2004-08-13 [PMID:15192095]
Abstract [show]
Structural evidence has demonstrated that P-glycoprotein (P-gp) undergoes considerable conformational changes during catalysis, and these alterations are important in drug interaction. Knowledge of which regions in P-gp undergo conformational alterations will provide vital information to elucidate the locations of drug binding sites and the mechanism of coupling. A number of investigations have implicated transmembrane segment six (TM6) in drug-P-gp interactions, and a cysteine-scanning mutagenesis approach was directed to this segment. Introduction of cysteine residues into TM6 did not disturb basal or drug-stimulated ATPase activity per se. Under basal conditions the hydrophobic probe coumarin maleimide readily labeled all introduced cysteine residues, whereas the hydrophilic fluorescein maleimide only labeled residue Cys-343. The amphiphilic BODIPY-maleimide displayed a more complex labeling profile. The extent of labeling with coumarin maleimide did not vary during the catalytic cycle, whereas fluorescein maleimide labeling of F343C was lost after nucleotide binding or hydrolysis. BODIPY-maleimide labeling was markedly altered during the catalytic cycle and indicated that the adenosine 5'-(beta,gamma-imino)triphosphate-bound and ADP/vanadate-trapped intermediates were conformationally distinct. Our data are reconciled with a recent atomic scale model of P-gp and are consistent with a tilting of TM6 in response to nucleotide binding and ATP hydrolysis.
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No. Sentence Comment
149 Mutant G324C, which contains a cysteine in an accessible extracellular loop, was used as a positive control for labeling.
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ABCB1 p.Gly324Cys 15192095:149:7
status: NEW153 Nonspecific labeling of Cys-less P-gp by FM was 4 Ϯ 1% (Fig. 2a), whereas G324C labeled to 100% with a half-life of 12 Ϯ 1 min (data not shown).
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ABCB1 p.Gly324Cys 15192095:153:80
status: NEW155 In contrast, F343C in the native protein conformation was accessible to labeling with FM as adjudged by the 81 Ϯ 2% labeling extent (Lext), which was characterized by a half-life (t1/2 ϭ 47 Ϯ 8 min) almost 4 times slower than observed for G324C.
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ABCB1 p.Gly324Cys 15192095:155:257
status: NEW158 The level of nonspecific interaction with the Cys-less P-gp was higher (Lext ϭ 25 Ϯ 4%), and G324C was also labeled by CM (Lext ϭ 102 Ϯ 4%), although the half-life (t1/2 ϭ 31 Ϯ 4 min) was almost 3 times slower than observed for FM.
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ABCB1 p.Gly324Cys 15192095:158:105
status: NEW162 Nonspecific interaction with Cys-less protein was 19 Ϯ 1% and G324C labeled fully (Lext ϭ 97 Ϯ 3%) with a half-life approximately double (t1/2 ϭ 23 Ϯ 2 min) that obtained for FM.
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ABCB1 p.Gly324Cys 15192095:162:68
status: NEW[hide] Cytosolic region of TM6 in P-glycoprotein: topogra... Biochemistry. 2008 Mar 25;47(12):3615-24. Epub 2008 Feb 28. Storm J, Modok S, O'Mara ML, Tieleman DP, Kerr ID, Callaghan R
Cytosolic region of TM6 in P-glycoprotein: topographical analysis and functional perturbation by site directed labeling.
Biochemistry. 2008 Mar 25;47(12):3615-24. Epub 2008 Feb 28., 2008-03-25 [PMID:18303860]
Abstract [show]
Reduced intracellular drug accumulation due to the activity of the drug efflux pump ABC (B1) is a major mechanism in the resistance of cancer cells to chemotherapy. ABC (B1) is a poly specific transporter, and the molecular mechanism of its complex translocation process remains to be elucidated. To understand the process will require information on the regions involved in drug binding and those that couple this event to nucleotide hydrolysis. The present investigation focuses on the cytosolic region of transmembrane helix 6 (TM6), which has been widely attributed with a central role in the translocation process. A series of ABC (B1) isoforms containing a unique cysteine within TM6 was constructed and the resultant proteins purified and reconstituted. Accessibility of the cysteines to covalent modification by maleimide reagents was measured for the basal, ATP bound and vanadate trapped conformations of each isoform. Residues at the two extremes of the TM6 region examined (amino acids 344 to 360) were considerably more accessible than the central segment, the latter of which also failed to undergo significant conformational changes during the catalytic cycle. Covalent modification of the cytosolic segment of TM6 did, however, attenuate drug stimulation of ATP hydrolysis and demonstrates an important role for this segment in coupling drug binding to ATP hydrolysis during translocation.
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No. Sentence Comment
115 The positive control lane refers to labeling of the G324C isoform for 300 min and was assigned a value of 100%.
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ABCB1 p.Gly324Cys 18303860:115:52
status: NEW137 The "+ve" lane refers to labeling of the G324C isoform for 300 min (b) The SDS-PAGE data was quantified using densitometric analysis, and the values were expressed as a percentage of that obtained for the G324C isoform.
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ABCB1 p.Gly324Cys 18303860:137:41
status: NEWX
ABCB1 p.Gly324Cys 18303860:137:205
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
47 Labelling was time dependent during the 300 min incubation, and the extents of labelling were quantified in comparison with that found with cysteine-less ABCB1 and the G324C isoform.
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ABCB1 p.Gly324Cys 20731718:47:168
status: NEW48 The G324C mutant was assigned as the positive control and given a value of 100%, as this residue is located on an external loop and is freely accessible to each of the probes used [25,28].
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ABCB1 p.Gly324Cys 20731718:48:4
status: NEW49 Furthermore, the complete labelling of the G324C mutant with the zwitterionic and hydrophilic probes BODIPY maleimide (BM) and fluorescein maleimide (FM), respectively, demonstrated that the protein was not preferentially oriented in one direction within the proteoliposomes.
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ABCB1 p.Gly324Cys 20731718:49:43
status: NEW50 Consequently, labelling of TM12 isoforms was determined as a percentage of G324C labelling, as outlined in Experimental procedures.
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ABCB1 p.Gly324Cys 20731718:50:75
status: NEW74 Lane (vii) contains the G324C isoform, which has been assigned a 100% value for labelling with BM.
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ABCB1 p.Gly324Cys 20731718:74:24
status: NEW103 These were then expressed as a percentage of the maximal extent of G324C labelling.
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ABCB1 p.Gly324Cys 20731718:103:67
status: NEW104 The degree of labelling (% of G324C level) was plotted as a function of time (min) and fitted with an exponential reaction curve, using nonlinear least squares regression.
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ABCB1 p.Gly324Cys 20731718:104:30
status: NEW136 The Lext for labelling is expressed as the fraction of specific labelling of single-cysteine isoforms over the specific labelling of the G324C positive control.
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ABCB1 p.Gly324Cys 20731718:136:137
status: NEW257 The G324C mutation, located on a freely accessible extracellular loop, has previously been demonstrated to be freely accessible to each maleimide probe [28]; labelling of the isoform containing this mutation was therefore assigned the value of 100% after 300 min.
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ABCB1 p.Gly324Cys 20731718:257:4
status: NEW258 Both the cysteine-less and G324C isoforms were incubated with 10 lm probe for 300 min and treated identically to the other isoforms.
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ABCB1 p.Gly324Cys 20731718:258:27
status: NEW259 The extent of labelling for each single-cysteine mutant was therefore determined by comparison with G324C.
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ABCB1 p.Gly324Cys 20731718:259:100
status: NEW261 The propensity for labelling was calculated with the following equation: L ¼ Liso À Lcys À Á L324C À Lcys À Á 100 where L is extent of labelling (%), Liso is the extent of isoform labelling, Lcys is labelling of the cysteine-less isoform, and L324C is labelling of the G324C isoform.
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ABCB1 p.Gly324Cys 20731718:261:307
status: NEW