ABCB1 p.Ile299Cys
| Predicted by SNAP2: | A: N (57%), C: N (61%), D: D (71%), E: D (71%), F: N (78%), G: D (63%), H: D (63%), K: D (75%), L: N (87%), M: N (93%), N: D (53%), P: D (71%), Q: N (57%), R: D (71%), S: N (61%), T: N (72%), V: N (78%), W: D (66%), Y: D (59%), |
| Predicted by PROVEAN: | A: N, C: N, D: D, E: D, F: N, G: D, H: D, K: N, L: N, M: N, N: D, P: D, Q: N, R: D, S: N, T: N, V: N, W: N, Y: N, |
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[hide] The packing of the transmembrane segments of human... J Biol Chem. 2000 Feb 25;275(8):5253-6. Loo TW, Clarke DM
The packing of the transmembrane segments of human multidrug resistance P-glycoprotein is revealed by disulfide cross-linking analysis.
J Biol Chem. 2000 Feb 25;275(8):5253-6., 2000-02-25 [PMID:10681495]
Abstract [show]
Residues from several transmembrane (TM) segments of P-glycoprotein (P-gp) likely form the drug-binding site(s). To determine the organization of the TM segments, pairs of cysteine residues were introduced into the predicted TM segments of a Cys-less P-gp, and the mutant protein was subjected to oxidative cross-linking. In SDS gels, the cross-linked product migrated with a slower mobility than the native protein. The cross-linked products were not detected in the presence of dithiothreitol. Cross-linking was observed in 12 of 125 mutants. The pattern of cross-linking suggested that TM6 is close to TMs 10, 11, and 12, while TM12 is close to TMs 4, 5, and 6. In some mutants the presence of drug substrate colchicine, verapamil, cyclosporin A, or vinblastine either enhanced or inhibited cross-linking. Cross-linking was inhibited in the presence of ATP plus vanadate. These results suggest that the TM segments critical for drug binding must be close to each other and exhibit different conformational changes in response to binding of drug substrate or vanadate trapping of nucleotide. Based on these results, we propose a model for the arrangement of the TM segments.
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None has been submitted yet.
No. Sentence Comment
65 Twelve of the 125 P-gp mutants (TM4/TM12 constructs L227C/S993C, V231C/S993C, W232C/S993C, A233C/S993C, I235C/S993C, and L236C/S993C; TM5/TM12 constructs A295C/S993C and I299C/S993C; TM10/TM6 constructs V874C/P350C, E875C/ P350C, and M876C/P350C; and TM11/TM6 construct G939C/ P350C), however, had slower mobilities in SDS-PAGE after treatment with oxidant.
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ABCB1 p.Ile299Cys 10681495:65:170
status: NEW77 In these cross-linking experiments, the amount of oxidant was lowered by 10-fold (0.2 mM), and the minimum temperature required to induce cross-TABLE I Cross-linking analysis of P-gp Cross-linking of S993C (TM12) with residues in the following TM: TM1 TM2 TM3 TM4 TM5 M51C -a Y130C - G185C - G226C - I293C - V52C - I131C - I186C - L227C ϩb T294C - V53C - Q132C - G187C - S228C - A295C ϩ G54C - V133C - D188C - A229C - N296C - T55C - S134C - K189C - A230C - I297C - L56C - F135C - I190C - V231C ϩ S298C - A57C - W136C - G191C - W232C ϩ I299C ϩ A58C - C137C - M192C - A233C ϩ G300C - I59C - L138C - F193C - K234C - A301C - I60C - A139C - F194C - I235C ϩ A302C - H61C - A140C - Q195C - L236C ϩ F303C - G141C - S196C - S237C - L304C - Cross-linking of P350C (TM6) with residues in the following TM: TM7 TM8 TM9 TM10 TM11 F711C - F770C - A828C - I867C - A935C - V712C - F771C - I829C - I868C - H936C - V713C - L772C - G830C - A869C - I937C - G714C - Q773C - S831C - I870C - F938C - V715C - G774C - R832C - A871C - G939C ϩ F716C - F775C - L833C - G872C - I940C - C717C - T776C - A834C - V873C - T941C - A718C - F777C - V835C - V874C ϩ F942C - I719C - G778C - I836C - E875C ϩ S943C - I720C - K779C - T837C - M876C ϩ F944C - N721C - A780C - Q838C - K877C - T945C - G722C - G781C - N839C - M878C - Q946C - G723C - E782C - I840C - L879C - A947C - I783C - a -, no cross-linked product detected in SDS-PAGE. b ϩ, cross-linked product detected in SDS-PAGE.
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ABCB1 p.Ile299Cys 10681495:77:559
status: NEW99 Mutants L227C/S993C, V231C/ S993C, W232C/S993C, A233C/S993C, I235C/S993C, L236C/ S993C, A295C/S993C, I299C/S993C, V874C/P350C, E875C/ P350C, M876C/P350C, and G939C/P350C were inhibited by 81, 88, 90, 89, 93, 81, 78, 87, 87, 77, 70, and 78%, respectively.
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ABCB1 p.Ile299Cys 10681495:99:101
status: NEW118 TABLE II Minimum temperature required for cross-linking Residues TM segments 4 °C 21 °C 37 °C L227C/S993C 4/12 -a - ϩ V231C/S993C 4/12 - ϩ ϩ W232C/S993C 4/12 - ϩ ϩ A233C/S993C 4/12 ϩb ϩ ϩ I235C/S993C 4/12 ϩ ϩ ϩ L236C/S993C 4/12 ϩ ϩ ϩ A295C/S993C 5/12 - ϩ ϩ I299C/S993C 5/12 ϩ ϩ ϩ V874C/P350C 10/6 - ϩ ϩ E875C/P350C 10/6 - - ϩ M876C/P350C 10/6 - ϩ ϩ G939C/P350C 11/6 - ϩ ϩ a -, no cross-linked product detected in SDS-PAGE. b ϩ, cross-linked product detected in SDS-PAGE.
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ABCB1 p.Ile299Cys 10681495:118:368
status: NEW[hide] Disulfide cross-linking analysis shows that transm... J Biol Chem. 2004 Feb 27;279(9):7692-7. Epub 2003 Dec 10. Loo TW, Bartlett MC, Clarke DM
Disulfide cross-linking analysis shows that transmembrane segments 5 and 8 of human P-glycoprotein are close together on the cytoplasmic side of the membrane.
J Biol Chem. 2004 Feb 27;279(9):7692-7. Epub 2003 Dec 10., 2004-02-27 [PMID:14670948]
Abstract [show]
Human P-glycoprotein (P-gp) transports a wide variety of structurally diverse compounds out of the cell. Knowledge about the packing of the transmembrane (TM) segments is essential for understanding the mechanism of drug recognition and transport. We used cysteine-scanning mutagenesis and disulfide cross-linking analysis to determine which TM segment in the COOH half of P-gp was close to TMs 5 and 6 since these segments in the NH(2) half are important for drug binding. An active Cys-less P-gp mutant cDNA was used to generate 240 double cysteine mutants that contained 1 cysteine in TMs 5 or 6 and another in TMs 7 or 8. The mutants were subjected to oxidative cross-linking analysis. No disulfide cross-linking was observed in the 140 TM6/TM7 or TM6/TM8 mutants. By contrast, cross-linking was detected in several P-gp TM5/TM8 mutants. At 4 degrees C, when thermal motion is low, P-gp mutants N296C(TM5)/G774C(TM8), I299C(TM5)/F770C(TM8), I299C(TM5)/G774C(TM8), and G300C(TM5)/F770C(TM8) showed extensive cross-linking with oxidant. These mutants retained drug-stimulated ATPase activity, but their activities were inhibited after treatment with oxidant. Similarly, disulfide cross-linking was inhibited by vanadate trapping of nucleotide. These results indicate that significant conformational changes must occur between TMs 5 and 8 during ATP hydrolysis. We revised the rotational symmetry model for TM packing based on our results and by comparison to the crystal structure of MsbA (Chang, G. (2003) J. Mol. Biol. 330, 419-430) such that TM5 is adjacent to TM8, TM2 is adjacent to TM11, and TMs 1 and 7 are next to TMs 6 and 12, respectively.
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No. Sentence Comment
101 Eleven mutants (I293C/F775C, N296C/F770C, N296C/G774C, I297C/F771C, I297C/G774C, I299C/F770C, I299C/G774C, G300C/F767C, G300C/F770C, G300C/F771C, and G300C/G774C) showed relatively strong (Ͼ50%) cross-linking (Table I).
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ABCB1 p.Ile299Cys 14670948:101:81
status: NEWX
ABCB1 p.Ile299Cys 14670948:101:94
status: NEW110 The eleven mutants (I293C/F775C, N296C/F770C, N296C/G774C, I297C/F771C, I297C/G774C, I299C/F770C, I299C/G774C, G300C/F767C, G300C/F770C, G300C/F771C, and G300C/G774C) that showed relatively strong cross-linking at 37 °C were subjected to cross-linking at 22 and 4 °C.
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ABCB1 p.Ile299Cys 14670948:110:85
status: NEWX
ABCB1 p.Ile299Cys 14670948:110:98
status: NEW111 Only four mutants (N296C/G774C, I299C/F770C, I299C/G774C, and G300C/F770C) still showed cross-linking at 22 and 4 °C.
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ABCB1 p.Ile299Cys 14670948:111:32
status: NEWX
ABCB1 p.Ile299Cys 14670948:111:45
status: NEW112 Mutants I293C/F775C, N296C/ F770C, I297C/G774C, G300C/F771C, and G300C/G774C were cross-linked only at 22 °C, whereas mutants I297C/F771C and G300C/F767C showed no cross-linking at either 22 or 4 °C. Fig. 3 shows the temperature-dependent cross-linking of mutants N296C/G774C, I299C/F770C, I299C/G774C, and G300C/ F770C.
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ABCB1 p.Ile299Cys 14670948:112:287
status: NEWX
ABCB1 p.Ile299Cys 14670948:112:300
status: NEW115 Mutants N296C/G774C, I299C/F770C, I299C/G774C, and G300C/F770C were selected for further analysis because they were cross-linked at 37, 22, and 4 °C.
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ABCB1 p.Ile299Cys 14670948:115:21
status: NEWX
ABCB1 p.Ile299Cys 14670948:115:34
status: NEW117 By contrast, no cross-linked product was detected in the single cysteine mutants, N296C, I299C, G300C, F770C, and G774C, when oxidant was added at these temperatures (data not shown).
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ABCB1 p.Ile299Cys 14670948:117:89
status: NEW118 To test if the mutants N296C/G774C, I299C/F770C, I299C/ G774C, and G300C/F770C retained the ability to interact with drug substrates, they were expressed in HEK 293 cells, isolated by nickel-chelate chromatography, mixed with lipid, and assayed for drug-stimulated ATPase activity.
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ABCB1 p.Ile299Cys 14670948:118:36
status: NEWX
ABCB1 p.Ile299Cys 14670948:118:49
status: NEW123 Mutant I299C/F770C had about the same amount of ATPase activity as Cys-less P-gp.
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ABCB1 p.Ile299Cys 14670948:123:7
status: NEW124 The verapamiland demecolcine-stimulated ATPase activities rel- TABLE I Cross-linking between residues in TMs 5 and 8 TM5 TM 8 F767C I768C T769C F770C F771C L772C Q773C G774C F775C T776C I293C -a - - - - - - - ϩϩb - T294C - - - - - - - - - - A295C - - - - - - - ϩ - - N296C - - - ϩϩb ϩ - ϩ ϩϩc ϩ - I297C - - - ϩd ϩϩ - - ϩϩb - - S298C - - - - - - - - - - I299C - - - ϩϩc - - - ϩϩc - - G300C ϩϩe - - ϩϩc ϩϩb - - ϩϩb ϩ - A301C - - - - - - - - - - A302C - - - - - - - - - - a No cross-linked product detected in SDS-PAGE gels at 37 °C. b Cross-linked product was also detected at 22 °C. c Cross-linked product was also detected at 22 °C and at 4 °C. d Relatively weak cross-linking (Ͻ50% of P-gp cross-linked) at 37 °C. e Relatively strong cross-linking (Ͼ50% of P-gp cross-linked) at 37 °C. FIG. 2.
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ABCB1 p.Ile299Cys 14670948:124:443
status: NEW130 ative to that of Cys-less P-gp were 65 and 62%, 68 and 51%, and 57 and 79% for mutants N296C/G774C, I299C/G774C, and G300C/F770C, respectively.
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ABCB1 p.Ile299Cys 14670948:130:100
status: NEW131 We then tested whether cross-linking affected the verapamil-stimulated ATPase activities of mutants N296C/G774C, I299C/ F770C, I299C/G774C, and G300C/F770C.
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ABCB1 p.Ile299Cys 14670948:131:113
status: NEWX
ABCB1 p.Ile299Cys 14670948:131:127
status: NEW134 By contrast, the verapamil-stimulated ATPase activities of mutants N296C/G774C, I299C/ F770C, I299C/G774C, and G300C/F770C were inhibited by 60-80% after treatment with oxidant. These results suggest that cross-linking inhibits conformational changes in P-gp during ATP hydrolysis (47, 50).
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ABCB1 p.Ile299Cys 14670948:134:80
status: NEWX
ABCB1 p.Ile299Cys 14670948:134:94
status: NEW135 Because there is evidence that TM5 lines the drug binding pocket of P-gp (44), we tested whether drug substrates that stimulate (demecolcine and verapamil) or inhibit (cyclosporin A) (29) ATPase activity would affect the cross-linking pattern observed in mutants N296C/G774C, I299C/F770C, I299C/ G774C, and G300C/F770C.
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ABCB1 p.Ile299Cys 14670948:135:276
status: NEWX
ABCB1 p.Ile299Cys 14670948:135:289
status: NEW138 Mutant I299C/F770C showed a small decrease in cross-linking with cyclosporin A and demecolcine, whereas mutant I299C/ G774C was only slightly affected by the drug substrates.
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ABCB1 p.Ile299Cys 14670948:138:7
status: NEWX
ABCB1 p.Ile299Cys 14670948:138:111
status: NEW147 Accordingly, the effect of vanadate trapping on cross-linking of mutants N296C/G774C, I299C/ F770C, I299C/G774C, and G300C/F770C was examined.
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ABCB1 p.Ile299Cys 14670948:147:86
status: NEWX
ABCB1 p.Ile299Cys 14670948:147:100
status: NEW151 Effect of temperature on cross-linking. Membranes were prepared from HEK 293 cells expressing P-gp mutants N296C/G774C, I299C/F770C, I299C/G774C, G300C/F770C, or N296C/F770C.
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ABCB1 p.Ile299Cys 14670948:151:120
status: NEWX
ABCB1 p.Ile299Cys 14670948:151:133
status: NEW157 Histidine-tagged Cys-less (C-less) P-gp and mutants N296C/ G774C, I299C/F770C, I299C/G774C, or G300C/F770C were isolated by nickel-chelate chromatography.
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ABCB1 p.Ile299Cys 14670948:157:66
status: NEWX
ABCB1 p.Ile299Cys 14670948:157:79
status: NEW162 Effect of drug substrates on cross-linking. Membranes were prepared from HEK 293 cells expressing P-gp mutants N296C/ G774C, I299C/F770C, I299C/G774C, or G300C/F770C.
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ABCB1 p.Ile299Cys 14670948:162:125
status: NEWX
ABCB1 p.Ile299Cys 14670948:162:138
status: NEW191 The model may explain why the presence of drug substrates had relatively little effect on cross-linking of mutants N296C/ G774C, I299C/F770C, I299C/G774C, and G300C/F770C.
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ABCB1 p.Ile299Cys 14670948:191:129
status: NEWX
ABCB1 p.Ile299Cys 14670948:191:142
status: NEW197 Membranes from HEK 293 cells expressing P-gp mutants N296C/G774C, I299C/F770C, I299C/G774C, or G300C/F770C were preincubated at 37 °C for 10 min in the presence (ϩ) or absence (-) of ATP and MgCl2 plus sodium vanadate (ATP/VO4).
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ABCB1 p.Ile299Cys 14670948:197:66
status: NEWX
ABCB1 p.Ile299Cys 14670948:197:79
status: NEW211 For example, vanadate trapping of nucleotide nearly abolished cross-linking in mutants N296C/ G774C, I299C/F770C, I299C/G774C, and G300C/F770C (Fig. 6), although cross-linking inhibited drug-stimulated ATPase activity.
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ABCB1 p.Ile299Cys 14670948:211:101
status: NEWX
ABCB1 p.Ile299Cys 14670948:211:114
status: NEW