ABCB1 p.Gln535Cys
| Predicted by SNAP2: | A: D (80%), C: D (75%), D: D (85%), E: D (75%), F: D (85%), G: D (85%), H: D (80%), I: D (85%), K: D (91%), L: D (85%), M: D (80%), N: D (85%), P: D (91%), R: D (91%), S: D (80%), T: D (85%), V: D (80%), W: D (85%), Y: D (80%), |
| 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] 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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89 CP, copper phenanthroline TABLE I Cross-linking between residues in the NBD1 signature sequence and in the NBD2 Walker A site L531C (21%)a S532C (38%) G533C (91%) G534C (4%) Q535C (0%) 4 °C 21 °C 37 °C 4 °C 21 °C 37 °C 4 °C 21 °C 37 °C 4 °C 21 °C 37 °C 4 °C 21 °C 37 °C G1070C (0%) -b - - - - - - - - - - - - - - S1071C (85%) - - *c - - - - - - - - ϩ - - ϩ S1072C (23%) - ϩc ϩϩd - ϩϩ ϩϩ - - ϩ - - ϩ - - - G1073C (4%) ϩ ϩϩ ϩϩ ϩ ϩϩ ϩϩ - ϩ ϩϩ - - - - - - C1074 (103%) ** ** **d,e - * * - - * - - - - - - G1075C (0%) - - ϩϩ - - ϩ - - - - - - - - - K1076C (12%) - - * - - - - - - - - - - - - S1077C (0%) - - - - - - - - - - - - - - - a Activity of the single cysteine mutant relative to Cys-less P-gp. b No cross-linked product detected in SDS-PAGE. c Relatively weak cross-linking (Ͻ50% of P-gp cross-linked).
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ABCB1 p.Gln535Cys 12226074:89:174
status: NEW[hide] The power of the pump: mechanisms of action of P-g... Eur J Pharm Sci. 2006 Apr;27(5):392-400. Epub 2005 Dec 13. Ambudkar SV, Kim IW, Sauna ZE
The power of the pump: mechanisms of action of P-glycoprotein (ABCB1).
Eur J Pharm Sci. 2006 Apr;27(5):392-400. Epub 2005 Dec 13., [PMID:16352426]
Abstract [show]
Members of the superfamily of ATP-binding cassette (ABC) transporters mediate the movement of a variety of substrates including simple ions, complex lipids and xenobiotics. At least 18 ABC transport proteins are associated with disease conditions. P-glycoprotein (Pgp, ABCB1) is the archetypical mammalian ABC transport protein and its mechanism of action has received considerable attention. There is strong biochemical evidence that Pgp moves molecular cargo against a concentration gradient using the energy of ATP hydrolysis. However, the molecular details of how the energy of ATP hydrolysis is coupled to transport remain in dispute and it has not been possible to reconcile the data from various laboratories into a single model. The functional unit of Pgp consists of two nucleotide binding domains (NBDs) and two trans-membrane domains which are involved in the transport of drug substrates. Considerable progress has been made in recent years in characterizing these functionally and spatially distinct domains of Pgp. In addition, our understanding of the domains has been augmented by the resolution of structures of several non-mammalian ABC proteins. This review considers: (i) the role of specific conserved amino acids in ATP hydrolysis mediated by Pgp; (ii) emerging insights into the dimensions of the drug binding pocket and the interactions between Pgp and the transport substrates and (iii) our current understanding of the mechanisms of coupling between energy derived from ATP binding and/or hydrolysis and efflux of drug substrates.
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52 Between the Walker A and B sequences is found a linker peptide with the sequence LSGGQ, also known as the C-region or ABC signature sequence, as it is the hallmark of Table 1 - Summary of mutational analysis of conserved residues in nucleotide-binding domains of Pgp Domain Source Residue number Function Reference NBD1 NBD2 A-loop Human Y401A Y1044A No ATP binding/hydrolysis Kim et al. (submitted for publication) Walker A Mouse K429N K1072N Normal ATP binding but no hydrolysis Azzaria et al. (1989) G431A G1073A Human C431 C1074 ATP protects from modification by N-ethylmaleimide Loo and Clarke (1995) Disulfide bond formation between Walker A domains of both NBDs Urbatsch et al. (2001) Human K433M K1076M Decreased ATP-binding Muller et al. (1996) No ATP hydrolysis Szakacs et al. (2000) No vanadate-trapping, but aluminum and beryllium fluoride-induced trapping normal Q-loop Mouse Q471 Q1114 Not essential for ATP hydrolysis but may be involved in communication with drug-substrate sites Urbatsch et al. (2000a) LSGGQ or linker peptide or signature motif Mouse S528A S1173A Normal ATP binding but no hydrolysis Tombline et al. (2004a) Human S532R Decreased cell surface expression Hoof et al. (1994) Human G534C G1179C No ATP hydrolysis Loo et al. (2002) Human G534D Decreased cell surface expression Hoof et al. (1994) No drug resistance Normal cell surface expression Bakos et al. (1997) No ATP hydrolysis Human G534D/V G1179D Interdomain communication Szakacs et al. (2001) Human Q535C Q1180C No ATP hydrolysis Loo et al. (2002) Human K536Q Decreased drug resistance Hoof et al. (1994) LSGGQ or linker peptide or signature motif Human K536R Increased colchicine resistance (normal ATP hydrolysis?)
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ABCB1 p.Gln535Cys 16352426:52:1493
status: NEW