ABCMdb

PMID: 12226074

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 [PubMed]

Sentences

No. Mutations Sentence Comment

50 ABCB1 p.Ser1072Cys ABCB1 p.Leu531Cys ABCB1 p.Leu531Cys ABCB1 p.Leu531Cys ABCB1 p.Gly1070Cys The cross-linking results of three mutants, L531C/G1070C, L531C/S1072C, and L531C/C1074, are shown in Fig. 1. Login to comment 51 ABCB1 p.Leu531Cys ABCB1 p.Gly1070Cys No cross-linked product was detected in mutant L531C/G1070C at any temperature. Login to comment 52 ABCB1 p.Ser1072Cys ABCB1 p.Leu531Cys In mutant L531C/S1072C, no cross-linked product was detected when treated with oxidant at 4 °C. Login to comment 56 ABCB1 p.Leu531Cys In mutant L531C/ C1074, cross-linked product was detected at all three temperatures. Login to comment 58 ABCB1 p.Leu531Cys ABCB1 p.Leu531Cys ABCB1 p.Leu531Cys ABCB1 p.Gly1073Cys ABCB1 p.Gly1073Cys ABCB1 p.Gly1073Cys The complete cross-linking results between the cysteines in the NBD1 terminal signature sequence (531 LSGGQ535 ) and the cysteines in the NBD2 Walker A site (1070 GSSGCGKS1077 ) are shown in Table I. Residues G1073C and C1074 in the NBD2 Walker A site and L531C and S532C in the NBD1 signature sequence appear to be closest, since mutants L531C/G1073C, L531C/C1074, and S532C/G1073C were cross-linked when treated with oxidant at 4 °C. Login to comment 59 ABCB1 p.Ser1072Cys ABCB1 p.Ser1072Cys ABCB1 p.Leu531Cys ABCB1 p.Gly1073Cys ABCB1 p.Gly533Cys The cysteines in other mutants (e.g. L531C/S1072C, S532/S1072C, and G533C/G1073C) must be further apart, since cross-linked product was only observed at either 21 or 37 °C. Login to comment 66 ABCB1 p.Leu531Cys ABCB1 p.Leu531Cys ABCB1 p.Gly1073Cys Representative cross-linking results of two (NBD1 signature/NBD2 Walker A) mutants (L531C/G1073C and L531C/C1074) are shown in Fig. 2. Login to comment 69 ABCB1 p.Leu531Cys Cross-linking of mutant L531C/C1074 was almost completely inhibited by vanadate trapping of nucleotide. Login to comment 70 ABCB1 p.Leu531Cys ABCB1 p.Leu531Cys ABCB1 p.Gly533Cys ABCB1 p.Ser1071Cys ABCB1 p.Lys1076Cys Similar results were observed in mutants L531C/ S1071C, S532C/C1074, G533C/C1074C, and L531C/K1076C (Table I). Login to comment 71 ABCB1 p.Leu531Cys ABCB1 p.Leu531Cys ABCB1 p.Leu531Cys ABCB1 p.Gly533Cys ABCB1 p.Ser1071Cys ABCB1 p.Lys1076Cys Mutants L531C/C1074, L531C/S1071C, S532C/ C1074, G533C/C1074, and L531C/K1076C had verapamil-stimulated ATPase activities of 22, 22, 30, 90, and 11% respectively, relative to Cys-less P-gp. Login to comment 72 ABCB1 p.Leu531Cys ABCB1 p.Gly1073Cys The other mutants (Table I) such as L531C/G1073C (Fig. 2) showed no detectable inhibition of cross-linking when preincubated with ATP plus vanadate. Login to comment 74 ABCB1 p.Leu531Cys ABCB1 p.Gly1073Cys This appears to be the case for mutants such as L531C/G1073C that showed no inhibition of cross-linking after treatment with ATP plus vanadate. Login to comment 77 ABCB1 p.Ser434Cys ABCB1 p.Ser434Cys ABCB1 p.Gly427Cys ABCB1 p.Gly427Cys ABCB1 p.Asn428Cys ABCB1 p.Asn428Cys 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. Login to comment 79 ABCB1 p.Leu1176Cys One mutant, L1176C/C431, was cross-linked at 4, 21, and 37 °C. Login to comment 80 ABCB1 p.Leu1176Cys ABCB1 p.Leu1176Cys ABCB1 p.Leu1176Cys ABCB1 p.Gly432Cys ABCB1 p.Gly432Cys ABCB1 p.Ser1177Cys ABCB1 p.Ser1177Cys ABCB1 p.Ser1177Cys ABCB1 p.Ser1177Cys ABCB1 p.Gly1179Cys ABCB1 p.Gly430Cys ABCB1 p.Gly430Cys 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 82 ABCB1 p.Leu1176Cys Cross-linking of four mutants, L1176C/ FIG. 1. Login to comment 84 ABCB1 p.Ser1072Cys ABCB1 p.Leu531Cys ABCB1 p.Leu531Cys ABCB1 p.Leu531Cys ABCB1 p.Gly1070Cys Membranes were prepared from HEK 293 cells expressing mutants L531C/G1070C, L531C/ S1072C, or L531C/C1074. Login to comment 89 ABCB1 p.Ser1072Cys ABCB1 p.Leu531Cys ABCB1 p.Gly1070Cys ABCB1 p.Gly1073Cys ABCB1 p.Gly533Cys ABCB1 p.Ser1071Cys ABCB1 p.Lys1076Cys ABCB1 p.Ser1077Cys ABCB1 p.Gln535Cys ABCB1 p.Gly534Cys ABCB1 p.Gly1075Cys 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). Login to comment 92 ABCB1 p.Leu1176Cys ABCB1 p.Ser1177Cys ABCB1 p.Ser1177Cys S429C, S1177C/S429C, L1176C/C431, and S1177C/C431, was inhibited by preincubation with ATP plus vanadate (Table II). Login to comment 93 ABCB1 p.Leu1176Cys A representative blot of mutant L1176C/C431 is shown in Fig. 2. Login to comment 94 ABCB1 p.Leu1176Cys ABCB1 p.Leu1176Cys ABCB1 p.Gly432Cys ABCB1 p.Gly432Cys ABCB1 p.Ser1177Cys ABCB1 p.Ser1177Cys ABCB1 p.Gly430Cys ABCB1 p.Gly430Cys 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 105 ABCB1 p.Leu531Cys ABCB1 p.Leu1176Cys 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). Login to comment 106 ABCB1 p.Leu531Cys ABCB1 p.Leu1176Cys Fig. 3 shows that the activities of mutants L531C/C1074 and L1176C/C431 after treatment with oxidant were inhibited 82 and 72%, respectively. Login to comment 112 ABCB1 p.Leu531Cys ABCB1 p.Leu531Cys ABCB1 p.Gly1073Cys ABCB1 p.Leu1176Cys 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. Login to comment 118 ABCB1 p.Leu531Cys ABCB1 p.Leu1176Cys 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. Login to comment 124 ABCB1 p.Leu1176Cys ABCB1 p.Gly432Cys ABCB1 p.Ser1177Cys ABCB1 p.Gly1179Cys ABCB1 p.Gly430Cys ABCB1 p.Lys433Cys ABCB1 p.Gly1178Cys ABCB1 p.Gln1180Cys 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 131 ABCB1 p.Leu531Cys ABCB1 p.Gly1073Cys Hydrolysis of ATP was essential, since inhibition of cross-linking was not observed in the presence of the non-hydrolyzable ATP analog AMP.PNP (data not shown) or in the inactive mutants (e.g. mutant L531C/G1073C; Fig. 2). Login to comment