ABCB1 p.Leu1176Cys
| Predicted by SNAP2: | A: D (85%), C: D (75%), D: D (95%), E: D (91%), F: D (75%), G: D (91%), H: D (91%), I: D (71%), K: D (91%), M: D (59%), N: D (91%), P: D (91%), Q: D (91%), R: D (91%), S: D (91%), T: D (91%), V: D (71%), W: D (85%), Y: D (91%), |
| Predicted by PROVEAN: | A: D, C: D, D: D, E: D, F: D, G: D, H: D, I: N, K: D, M: N, N: D, P: D, Q: D, R: D, S: D, T: D, V: N, 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
79 One mutant, L1176C/C431, was cross-linked at 4, 21, and 37 °C.
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ABCB1 p.Leu1176Cys 12226074:79:12
status: NEW80 Mutants L1176C/S429C and L1176C/G432C were cross-linked only at 21 and 37 °C, while cross-linking of mutants L1176C/G430C, S1177C/S429C, S1177C/G430C, S1177C/ C431, S1177C/G432C, and G1179C/S429C was only observed at 37 °C.
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ABCB1 p.Leu1176Cys 12226074:80:8
status: NEWX
ABCB1 p.Leu1176Cys 12226074:80:25
status: NEWX
ABCB1 p.Leu1176Cys 12226074:80:114
status: NEW82 Cross-linking of four mutants, L1176C/ FIG. 1.
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ABCB1 p.Leu1176Cys 12226074:82:31
status: NEW92 S429C, S1177C/S429C, L1176C/C431, and S1177C/C431, was inhibited by preincubation with ATP plus vanadate (Table II).
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ABCB1 p.Leu1176Cys 12226074:92:21
status: NEW93 A representative blot of mutant L1176C/C431 is shown in Fig. 2.
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ABCB1 p.Leu1176Cys 12226074:93:32
status: NEW94 No inhibition of cross-linking by vanadate trapping was observed in mutants such as L1176C/G430C, L1176C/G432C, S1177C/G430C, or S1177C/G432C.
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ABCB1 p.Leu1176Cys 12226074:94:84
status: NEWX
ABCB1 p.Leu1176Cys 12226074:94:98
status: NEW105 Two mutants, L531C/C1074 and L1176C/C431, were selected for analysis; because these residues are found at identical positions when the two halves of P-gp are aligned, both mutants can be cross-linked with oxidant at 4 °C and both retained ATPase activity (22 and 41%, respectively, relative to Cys-less P-gp).
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ABCB1 p.Leu1176Cys 12226074:105:29
status: NEW106 Fig. 3 shows that the activities of mutants L531C/C1074 and L1176C/C431 after treatment with oxidant were inhibited 82 and 72%, respectively.
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ABCB1 p.Leu1176Cys 12226074:106:60
status: NEW112 Membranes prepared from HEK 293 cells expressing mutants L531C/ G1073C, L531C/C1074, or L1176C/C431 were preincubated for 10 min at 37 °C in the presence (ϩ) or absence (-) of ATP plus vanadate.
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ABCB1 p.Leu1176Cys 12226074:112:88
status: NEW118 Mutants L531C/C1074 and L1176C/C431 were isolated by nickel-chelate chromatography and treated with (ϩCP) or without (-CP) oxidant, copper phenanthroline (CP), for 15 min at 21 °C.
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ABCB1 p.Leu1176Cys 12226074:118:24
status: NEW124 Lines indicate residues cross-linked at 4 °C. TABLE II Cross-linking between residues in the NBD2 signature sequence and in the NBD1 Walker A site L1176C (53%)a S1177C (35%) G1178C (78%) G1179C (14%) Q1180C (23%) 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 S429C (38%) -b *c ** - - * - - - - - ϩ - - - G430C (12%) - - ϩϩd - - ϩ - - - - - - - - - C431 (100%) * ** **d,e - - * - - - - - - - - - G432C (0%) - ϩc ϩϩ - - ϩ - - - - - - - - - K433C (12%) - - - - - - - - - - - - - - - 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.Leu1176Cys 12226074:124:152
status: NEW[hide] Drug binding in human P-glycoprotein causes confor... J Biol Chem. 2003 Jan 17;278(3):1575-8. Epub 2002 Nov 5. Loo TW, Bartlett MC, Clarke DM
Drug binding in human P-glycoprotein causes conformational changes in both nucleotide-binding domains.
J Biol Chem. 2003 Jan 17;278(3):1575-8. Epub 2002 Nov 5., 2003-01-17 [PMID:12421806]
Abstract [show]
The human multidrug resistance P-glycoprotein (P-gp, ABCB1) uses ATP to transport many structurally diverse compounds out of the cell. It is an ABC transporter with two nucleotide-binding domains (NBDs) and two transmembrane domains (TMDs). Recently, we showed that the "LSGGQ" motif in one NBD ((531)LSGGQ(535) in NBD1; (1176)LSGGQ(1180) in NBD2) is adjacent to the "Walker A" sequence ((1070)GSSGCGKS(1077) in NBD2; (427)GNSGCGKS(434) in NBD1) in the other NBD (Loo, T. W., Bartlett, M. C., and Clarke, D. M. (2002) J. Biol. Chem. 277, 41303-41306). Drug substrates can stimulate or inhibit the ATPase activity of P-gp. Here, we report the effect of drug binding on cross-linking between the LSGGQ signature and Walker A sites (Cys(431)(NBD1)/C1176C(NBD2) and Cys(1074)(NBD2)/L531C(NBD1), respectively). Seven drug substrates (calcein-AM, demecolcine, cis(Z)-flupentixol, verapamil, cyclosporin A, Hoechst 33342, and trans(E)-flupentixol) were tested for their effect on oxidative cross-linking. Substrates that stimulated the ATPase activity of P-gp (calcein-AM, demecolcine, cis(Z)-flupentixol, and verapamil) increased the rate of cross-linking between Cys(431)(NBD1-Walker A)/C1176C(NBD2-LSGGQ) and between Cys(1074)(NBD2-Walker A)/L531C(NBD1-LSGGQ) when compared with cross-linking in the absence of drug substrate. By contrast, substrates that inhibited ATPase activity (cyclosporin A, Hoechst 33342, and trans(E)-flupentixol) decreased the rate of cross-linking. These results indicate that interaction between the LSGGQ motifs and Walker A sites must be essential for coupling drug binding to ATP hydrolysis. Drug binding in the transmembrane domains can induce long range conformational changes in the NBDs, such that compounds that stimulate or inhibit ATPase activity must decrease and increase, respectively, the distance between the Walker A and LSGGQ sequences.
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No. Sentence Comment
28 The other mutant (Cys431 / L1176C) contained the endogenous Cys431 in the NH2-terminal Walker A site (427 GNSGCGKS434 ) and another cysteine in the COOH-terminal 1176 LSGGQ1180 site.
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ABCB1 p.Leu1176Cys 12421806:28:27
status: NEW56 Fig. 1 shows that that cross-linking was almost complete in mutants L531C/Cys1074 and Cys431 /L1176C when treated with 0.5 mM copper phenanthroline for 15 min at 21 °C.
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ABCB1 p.Leu1176Cys 12421806:56:94
status: NEW63 Fig. 1 shows that inhibition of cross-linking in mutants L531C/Cys1074 and Cys431 /L1176C was observed only after treatment with vanadate plus Mg-ATP.
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ABCB1 p.Leu1176Cys 12421806:63:83
status: NEW70 Therefore, it is possible that the conformational changes in the TMDs may be monitored by changes in the cross-linking patterns in mutants L531C/Cys1074 and Cys431 /L1176C.
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ABCB1 p.Leu1176Cys 12421806:70:165
status: NEW73 These drug substrates were then tested on mutants L531C/Cys1074 and Cys431 / L1176C.
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ABCB1 p.Leu1176Cys 12421806:73:77
status: NEW77 Similar results were obtained with mutant Cys431 / L1176C (data not shown).
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ABCB1 p.Leu1176Cys 12421806:77:51
status: NEW83 Membranes were prepared from HEK 293 cells expressing mutants L531C/Cys1074 (L531C/C1074) or Cys431 /L1176C (C431/L1176C).
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ABCB1 p.Leu1176Cys 12421806:83:101
status: NEWX
ABCB1 p.Leu1176Cys 12421806:83:114
status: NEW101 We then tested the effect of drug substrates on cross-linking of mutant Cys431 /L1176C.
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ABCB1 p.Leu1176Cys 12421806:101:80
status: NEW106 The effect of substrates on mutants L531C/Cys1074 and Cys431 /L1176C were very similar.
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ABCB1 p.Leu1176Cys 12421806:106:62
status: NEW122 Effect of drug substrates on cross-linking of mutant Cys431 /L1176C.
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ABCB1 p.Leu1176Cys 12421806:122:61
status: NEW123 Cross-linking on membranes containing P-gp mutant Cys431 /L1176C were done as described in the legend to Fig. 3.
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ABCB1 p.Leu1176Cys 12421806:123:58
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
681 More recently, in the light of the NBD sandwich dimer, Loo et al. have demonstrated that the Walker-A cysteine can be cross-linked to a cysteine residue introduced within the opposite signature motif (L1176C, [19]), 10 Å apart (Cα-Cα, Table S1).
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ABCB1 p.Leu1176Cys 18035039:681:201
status: NEW676 More recently, in the light of the NBD sandwich dimer, Loo et al. have demonstrated that the Walker-A cysteine can be cross-linked to a cysteine residue introduced within the opposite signature motif (L1176C, [19]), 10 &#c5; apart (Cb1;-Cb1;, Table S1).
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ABCB1 p.Leu1176Cys 18035039:676:201
status: NEW