ABCMdb

ABCB1 p.Ser1177Cys

Predicted by SNAP2:	A: D (80%), C: D (80%), D: D (91%), E: D (91%), F: D (85%), G: D (91%), H: D (85%), I: D (91%), K: D (91%), L: D (91%), M: D (85%), N: D (91%), P: D (95%), Q: D (85%), R: D (91%), T: D (85%), V: D (85%), W: D (91%), Y: D (91%),
Predicted by PROVEAN:	A: N, C: D, D: D, E: D, F: D, G: D, H: D, I: D, K: D, L: D, M: D, N: D, P: D, Q: D, R: D, T: N, V: D, W: D, Y: D,

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Publications
[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
80 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. Login to comment
92 S429C, S1177C/S429C, L1176C/C431, and S1177C/C431, was inhibited by preincubation with ATP plus vanadate (Table II). Login to comment
94 No inhibition of cross-linking by vanadate trapping was observed in mutants such as L1176C/G430C, L1176C/G432C, S1177C/G430C, or S1177C/G432C. Login to comment
124 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). Login to comment