ABCB1 p.Gly427Cys
| Predicted by SNAP2: | A: D (91%), C: D (91%), D: D (95%), E: D (95%), F: D (95%), H: D (95%), I: D (95%), K: D (95%), L: D (95%), M: D (95%), N: D (91%), P: D (95%), Q: D (95%), R: D (95%), S: D (91%), T: D (95%), V: D (95%), W: D (95%), Y: D (95%), |
| 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] The "LSGGQ" motif in each nucleotide-binding domai... J Biol Chem. 2002 Nov 1;277(44):41303-6. Epub 2002 Sep 10. Loo TW, Bartlett MC, Clarke DM
The "LSGGQ" motif in each nucleotide-binding domain of human P-glycoprotein is adjacent to the opposing walker A sequence.
J Biol Chem. 2002 Nov 1;277(44):41303-6. Epub 2002 Sep 10., 2002-11-01 [PMID:12226074]
Abstract [show]
The human multidrug resistance P-glycoprotein (P-gp, ABCB1), a member of the ATP-binding cassette (ABC) family of transport proteins, actively transports many cytotoxic compounds out of the cell. ABC transporters have two nucleotide-binding domains (NBD) and two transmembrane domains. The presence of the conserved "signature" sequence (LSGGQ) in each NBD is a unique feature in these transporters. The function of the signature sequences is unknown. In this study, we tested whether the signature sequences ((531)LSGGQ(535) in NBD1; (1176)LSGGQ(1180) in NBD2) in P-gp are in close proximity to the opposing Walker A consensus nucleotide-binding sequences ((1070)GSSGCGKS(1077) in NBD2; (427)GNSGCGKS(434) in NBD1). Pairs of cysteines were introduced into a Cys-less P-gp at the signature and "Walker A" sites and the mutant P-gps were subjected to oxidative cross-linking. At 4 degrees C, when thermal motion is low, P-gp mutants (L531C(Signature)/C1074(Walker A) and C431(Walker A)/L1176C(Signature) were cross-linked. Cross-linking inhibited the drug-stimulated ATPase activities of these two mutants. Their activities were restored, however, after addition of the reducing agent, dithiothreitol. Vanadate trapping of nucleotide at the ATP-binding sites prevented cross-linking of the mutants. These results indicate that the signature sequences are adjacent to the opposing Walker A site. They likely participate in forming the ATP-binding sites and are displaced upon ATP hydrolysis. The resulting conformational change may be the signal responsible for coupling ATP hydrolysis to drug transport by inducing conformational changes in the transmembrane segments.
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No. Sentence Comment
77 Cross-linking involving residues G427C, N428C, and S434C was not done because the single cysteine mutants, G427C and S434C, showed little or no activity (Ͻ5%), while N428C was defectively processed and rapidly degraded.
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ABCB1 p.Gly427Cys 12226074:77:33
status: NEWX
ABCB1 p.Gly427Cys 12226074:77:107
status: NEW[hide] Correction of defective protein kinesis of human P... J Biol Chem. 1997 Jan 10;272(2):709-12. Loo TW, Clarke DM
Correction of defective protein kinesis of human P-glycoprotein mutants by substrates and modulators.
J Biol Chem. 1997 Jan 10;272(2):709-12., 1997-01-10 [PMID:8995353]
Abstract [show]
There is growing evidence that abnormal protein folding or trafficking (protein kinesis) leads to diseases. We have used P-glycoprotein as a model protein to develop strategies to overcome defects in protein kinesis. Misprocessed mutants of the human P-glycoprotein are retained in the endoplasmic reticulum as core-glycosylated biosynthetic intermediates and rapidly degraded. Synthesis of the mutant proteins in the presence of drug substrates or modulators such as capsaicin, cyclosporin, vinblastine, or verapamil, however, resulted in the appearance of a fully glycosylated and functional protein at the cell surface. These effects were dose-dependent and occurred within a few hours after the addition of substrate. The ability to facilitate processing of the misfolded mutants appeared to be independent of the cell lines used and location of the mutation. P-glycoproteins with mutations in transmembrane segments, extracellular or cytoplasmic loops, the nucleotide-binding domains, or the linker region were processed to the fully mature form in the presence of these substrates. These drug substrates or modulators acted as specific chemical chaperones for P-glycoprotein because they were ineffective on the deltaF508 mutant of cystic fibrosis transmembrane conductance regulator. Therefore, one possible strategy to prevent protein misfolding is to carry out synthesis in the presence of specific substrates or modulators of the protein.
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No. Sentence Comment
64 In addition to the mutants G268V and ⌬Y490, we were able to facilitate processing of P-glycoproteins with mutations in the predicted transmembrane segments (TM1, G54V; TM5, G300V; TM7, A718L; and TM9, A841L), in the extracellular loops between transmembrane segments (G854V), in the cytoplasmic loops (G251V and W803A), in the nucleotide-binding domains (G427C and S434C), and in the linker region connecting the two halves of the molecule (E707A) (data not shown).
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ABCB1 p.Gly427Cys 8995353:64:362
status: NEW[hide] Superfolding of the partially unfolded core-glycos... J Biol Chem. 1998 Jun 12;273(24):14671-4. Loo TW, Clarke DM
Superfolding of the partially unfolded core-glycosylated intermediate of human P-glycoprotein into the mature enzyme is promoted by substrate-induced transmembrane domain interactions.
J Biol Chem. 1998 Jun 12;273(24):14671-4., 1998-06-12 [PMID:9614062]
Abstract [show]
Misprocessed mutants of human P-glycoprotein accumulate as core-glycosylated intermediates in the endoplasmic reticulum and are rapidly degraded. Trypsin digestion was used to test for structural differences between mature and core-glycosylated forms of P-glycoprotein. We found that the core-glycosylated wild-type and mutant P-glycoproteins were both 100-fold more sensitive to trypsin compared with the mature form of the wild-type enzyme. This result suggested that the core-glycosylated forms of both wild-type and mutant P-glycoproteins have similar unfolded structures, whereas the mature enzyme is folded into a more compact structure. The core-glycosylated mutant P-glycoproteins could be converted to the mature trypsin-resistant form by synthesis in the presence of drug substrate. Addition of proteasome inhibitor MG-132 to stabilize the core-glycosylated intermediate resulted in the accumulation but not maturation of the mutant protein. Further analysis showed that the second transmembrane domain TMD2 also became more resistant to trypsin digestion only after coexpression with TMD1 in the presence of substrate. Taken together, these results suggest that simply stabilizing the core-glycosylated intermediate is not sufficient to promote maturation of the processing mutants and that drug substrates induce maturation by promoting superfolding of the transmembrane domains.
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No. Sentence Comment
48 Misprocessing mutations are located throughout the molecule; these include the transmembrane domains (e.g. A123L), intracellular (e.g. E243A) and extracellular (e.g. Y853C) loops, the linker region (e.g. E707A), and both nucleotide-binding domains (e.g. G427C, P1194A).
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ABCB1 p.Gly427Cys 9614062:48:254
status: NEW[hide] Structure-based interpretation of the mutagenesis ... Biochim Biophys Acta. 2008 Feb;1778(2):376-91. Epub 2007 Nov 1. Lawson J, O'Mara ML, Kerr ID
Structure-based interpretation of the mutagenesis database for the nucleotide binding domains of P-glycoprotein.
Biochim Biophys Acta. 2008 Feb;1778(2):376-91. Epub 2007 Nov 1., [PMID:18035039]
Abstract [show]
P-glycoprotein (P-gp) is the most intensively studied eukaryotic ATP binding cassette (ABC) transporter, due to its involvement in the multidrug resistance phenotype of a number of cancers. In common with most ABC transporters, P-gp is comprised of two transmembrane domains (TMDs) and two nucleotide binding domains (NBD), the latter coupling ATP hydrolysis with substrate transport (efflux in the case of P-gp). Biochemical investigations over the past twenty years have attempted to unlock mechanistic aspects of P-glycoprotein through scanning and site-directed mutagenesis of both the TMDs and the NBDs. Contemporaneously, crystallographers have elucidated the atomic structure of numerous ABC transporter NBDs, as well as the intact structure (i.e. NBDs and TMDs) of a distantly related ABC-exporter Sav1866. Significantly, the structure of P-gp remains unknown, and only low resolution electron microscopy data exists. Within the current manuscript we employ crystallographic data for homologous proteins, and a molecular model for P-gp, to perform a structural interpretation of the existing "mutagenesis database" for P-gp NBDs. Consequently, this will enable testable predictions to be made that will result in further in-roads into our understanding of this clinically important drug pump.
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No. Sentence Comment
679 Two studies have proposed that the Walker-A cysteine residues can be cross-linked to the opposite Walker-A motif [73,74], either through a direct disulphide between the two naturally occurring Walker-A cysteines [74] or between the Walker-A cysteine of NBD2 and a G427C Walker-A NBD1 mutation [73].
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ABCB1 p.Gly427Cys 18035039:679:264
status: NEW674 Two studies have proposed that the Walker-A cysteine residues can be cross-linked to the opposite Walker-A motif [73,74], either through a direct disulphide between the two naturally occurring Walker-A cysteines [74] or between the Walker-A cysteine of NBD2 and a G427C Walker-A NBD1 mutation [73].
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ABCB1 p.Gly427Cys 18035039:674:264
status: NEW