ABCB1 p.Leu443Cys
| Predicted by SNAP2: | A: D (59%), C: N (78%), D: D (75%), E: D (63%), F: N (97%), G: D (66%), H: D (59%), I: N (66%), K: D (71%), M: N (87%), N: D (53%), P: D (71%), Q: D (59%), R: D (66%), S: D (59%), T: D (53%), V: N (66%), W: D (53%), Y: N (66%), |
| Predicted by PROVEAN: | A: D, C: D, D: D, E: D, F: N, 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: N, Y: N, |
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[hide] The ATPase activity of the P-glycoprotein drug pum... J Biol Chem. 2012 Aug 3;287(32):26806-16. doi: 10.1074/jbc.M112.376202. Epub 2012 Jun 14. Loo TW, Bartlett MC, Detty MR, Clarke DM
The ATPase activity of the P-glycoprotein drug pump is highly activated when the N-terminal and central regions of the nucleotide-binding domains are linked closely together.
J Biol Chem. 2012 Aug 3;287(32):26806-16. doi: 10.1074/jbc.M112.376202. Epub 2012 Jun 14., [PMID:22700974]
Abstract [show]
The P-glycoprotein (P-gp, ABCB1) drug pump protects us from toxic compounds and confers multidrug resistance. Each of the homologous halves of P-gp is composed of a transmembrane domain (TMD) with 6 TM segments followed by a nucleotide-binding domain (NBD). The predicted drug- and ATP-binding sites reside at the interface between the TMDs and NBDs, respectively. Crystal structures and EM projection images suggest that the two halves of P-gp are separated by a central cavity that closes upon binding of nucleotide. Binding of drug substrates may induce further structural rearrangements because they stimulate ATPase activity. Here, we used disulfide cross-linking with short (8 A) or long (22 A) cross-linkers to identify domain-domain interactions that activate ATPase activity. It was found that cross-linking of cysteines that lie close to the LSGGQ (P517C) and Walker A (I1050C) sites of NBD1 and NBD2, respectively, as well as the cytoplasmic extensions of TM segments 3 (D177C or L175C) and 9 (N820C) with a short cross-linker activated ATPase activity over 10-fold. A pyrylium compound that inhibits ATPase activity blocked cross-linking at these sites. Cross-linking between the NBDs was not inhibited by tariquidar, a drug transport inhibitor that stimulates P-gp ATPase activity but is not transported. Cross-linking between extracellular cysteines (T333C/L975C) predicted to lock P-gp into a conformation that prevents close NBD association inhibited ATPase activity. The results suggest that trapping P-gp in a conformation in which the NBDs are closely associated likely mimics the structural rearrangements caused by binding of drug substrates that stimulate ATPase activity.
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No. Sentence Comment
106 The location of residues that were mutated to cysteine to test for the effect of cross-linking between NBD1 and NBD2 (P517C/I1050C), NBD1 and TMD2 (L443C/S909C), ICL1 and ICL3 (D177C/N820C), TM segments 2 and 11 (C137/A935C) or 6 and 12 (T333C/L975C) are shown.
X
ABCB1 p.Leu443Cys 22700974:106:148
status: NEW152 The ATPase activity of M17M cross-linked L175C/N820C could still be highly activated when assayed in the presence of verapamil.
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ABCB1 p.Leu443Cys 22700974:152:7
status: NEW154 ATPase Activity Is Not Activated when the Homologous Halves Are Cross-linked at Locations Predicted Not to Undergo Large Distance Changes during the Open to Closed Conformational Change-Mutant C137/A935C contains natural Cys-137 in TM segment 2 (TMD1) and the A935C mutation in TM segment 11 (TMD2).
X
ABCB1 p.Leu443Cys 22700974:154:63
status: NEWX
ABCB1 p.Leu443Cys 22700974:154:214
status: NEW156 Mutant L443C/S909C contains a cysteine (L443) in NBD1 and a cysteine (S909C) in the ICL4 loop that connects the cytoplasmic extensions of TM segments 10 and 11.
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ABCB1 p.Leu443Cys 22700974:156:7
status: NEW158 In previous studies we showed that mutants C137/A935C (39) and L443C/ S909C (27) had verapamil-stimulated ATPase activities similar to Cys-less P-gp.
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ABCB1 p.Leu443Cys 22700974:158:63
status: NEW159 Membranes prepared from cells expressing mutants C137/ A935C or L443C/S909C were treated with M4M cross-linker.
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ABCB1 p.Leu443Cys 22700974:159:64
status: NEW163 These results indicate that stimulation of ATPase activity observed in M4M cross-linked mutants P517C/I1050C (Fig. 2B), D177C/N820C, or L175C/N820C (Fig. 3C) was likely a specific effect of trapping the protein in the closed conformation.
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ABCB1 p.Leu443Cys 22700974:163:117
status: NEW166 A, membranes prepared from HEK 293 cells expressing the NBD1/TMD2 mutant L443C/ S909C or the TM2/TM11 mutant C137/A935C were treated with (ϩ) or without (-) the M4M cross-linker.
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ABCB1 p.Leu443Cys 22700974:166:73
status: NEW168 The positions of the cross-linked (X-link) and mature (170 kDa) P-gps are indicated. B, membranes expressing mutants L443C/ S909C or C137/A935C were treated with or without (None) M4M cross-linker and histidine-tagged P-gp isolated by nickel-chelate chromatography.
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ABCB1 p.Leu443Cys 22700974:168:117
status: NEW287 One explanation is that some movement is required at the C137/A935C and L443C/S909C interfaces during the catalytic cycle.
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ABCB1 p.Leu443Cys 22700974:287:72
status: NEW301 Direct cross-linking of cysteines in the mutants would trap C137/A935C or L443C/S909C at a distance of about 5.6 Å, the average distance between ␣ carbons in a disulfide bond (64).
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ABCB1 p.Leu443Cys 22700974:301:74
status: NEW304 Another plausible explanation as to why direct cross-linking of mutants C137/A935C or L443C/S909C inhibited activity is that cross-linking may have trapped the protein in an inactive conformation with the sites closer than normal.
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ABCB1 p.Leu443Cys 22700974:304:86
status: NEW104 The location of residues that were mutated to cysteine to test for the effect of cross-linking between NBD1 and NBD2 (P517C/I1050C), NBD1 and TMD2 (L443C/S909C), ICL1 and ICL3 (D177C/N820C), TM segments 2 and 11 (C137/A935C) or 6 and 12 (T333C/L975C) are shown.
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ABCB1 p.Leu443Cys 22700974:104:148
status: NEW161 A, membranes prepared from HEK 293 cells expressing the NBD1/TMD2 mutant L443C/ S909C or the TM2/TM11 mutant C137/A935C were treated with (af9;) or without (afa;) the M4M cross-linker.
X
ABCB1 p.Leu443Cys 22700974:161:73
status: NEW280 One explanation is that some movement is required at the C137/A935C and L443C/S909C interfaces during the catalytic cycle.
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ABCB1 p.Leu443Cys 22700974:280:72
status: NEW294 Direct cross-linking of cysteines in the mutants would trap C137/A935C or L443C/S909C at a distance of about 5.6 &#c5;, the average distance between ॷ carbons in a disulfide bond (64).
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ABCB1 p.Leu443Cys 22700974:294:74
status: NEW297 Another plausible explanation as to why direct cross-linking of mutants C137/A935C or L443C/S909C inhibited activity is that cross-linking may have trapped the protein in an inactive conformation with the sites closer than normal.
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ABCB1 p.Leu443Cys 22700974:297:86
status: NEW[hide] Human P-glycoprotein contains a greasy ball-and-so... J Biol Chem. 2013 Jul 12;288(28):20326-33. doi: 10.1074/jbc.M113.484550. Epub 2013 Jun 3. Loo TW, Bartlett MC, Clarke DM
Human P-glycoprotein contains a greasy ball-and-socket joint at the second transmission interface.
J Biol Chem. 2013 Jul 12;288(28):20326-33. doi: 10.1074/jbc.M113.484550. Epub 2013 Jun 3., [PMID:23733192]
Abstract [show]
The P-glycoprotein drug pump protects us from toxins. Drug-binding sites in the transmembrane (TM) domains (TMDs) are connected to the nucleotide-binding domains (NBDs) by intracellular helices (IHs). TMD-NBD cross-talk is a key step in the transport mechanism because drug binding stimulates ATP hydrolysis followed by drug efflux. Here, we tested whether the IHs are critical for maturation and TMD-NBD coupling by characterizing the effects of mutations to the IH1 and IH2 interfaces. Although IH1 mutations had little effect, most mutations at the IH2-NBD2 interface inhibited maturation or activity. For example, the F1086A mutation at the IH2-NBD2 interface abolished drug-stimulated ATPase activity. The mutant F1086A, however, retained the ability to bind ATP and drug substrates. The mutant was defective in mediating ATP-dependent conformational changes in the TMDs because binding of ATP no longer promoted cross-linking between cysteines located at the extracellular ends of TM segments 6 and 12. Replacement of Phe-1086 (in NBD2) with hydrophobic but not charged residues yielded active mutants. The activity of the F1086A mutant could be restored when the nearby residue Ala-266 (in IH2) was replaced with aromatic residues. These results suggest that Ala-266/Phe-1086 lies in a hydrophobic IH2-NBD2 "ball-and-socket" joint.
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No. Sentence Comment
76 Effect of F1086A on Vanadate Trapping-The double cysteine mutant L443C/S909C was used to test whether the F1086A mutation inhibited vanadate trapping of nucleotide.
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ABCB1 p.Leu443Cys 23733192:76:65
status: NEW77 Vanadate trapping inhibits cross-linking of L443C/S909C P-gp (20).
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ABCB1 p.Leu443Cys 23733192:77:44
status: NEW78 Membranes prepared from cells expressing L443C/S909C or L443C/S909C/F1086A were incubated in the presence or absence of 5 mM MgATP plus 0.2 mM vanadate for 5 min at 37 &#b0;C. Samples were then treated with 1 mM copper phenanthroline for 15 min at 0 &#b0;C. The reactions were performed using a protein concentration of 0.4 mg/ml.
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ABCB1 p.Leu443Cys 23733192:78:41
status: NEWX
ABCB1 p.Leu443Cys 23733192:78:56
status: NEW111 By contrast, introduction of cysteine mutations at the equivalent sites between IH4 (S909C) and NBD1 (L443C) (Fig. 3, A and B) had no effect on activity (21).
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ABCB1 p.Leu443Cys 23733192:111:102
status: NEW141 E, membranes prepared from cells expressing L443C/S909C af9; F1086A were first treated with (af9;VO4) or without (afa;VO4) MgATP plus vanadate.
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ABCB1 p.Leu443Cys 23733192:141:44
status: NEW146 To test whether mutant F1086A retained the ability to bind and hydrolyze ATP, we tested whether vanadate trapping of ATP would still inhibit cross-linking of mutant F1086A/L443C/S909C.
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ABCB1 p.Leu443Cys 23733192:146:172
status: NEW[hide] The Transmission Interfaces Contribute Asymmetrica... J Biol Chem. 2015 Jul 3;290(27):16954-63. doi: 10.1074/jbc.M115.652602. Epub 2015 May 18. Loo TW, Clarke DM
The Transmission Interfaces Contribute Asymmetrically to the Assembly and Activity of Human P-glycoprotein.
J Biol Chem. 2015 Jul 3;290(27):16954-63. doi: 10.1074/jbc.M115.652602. Epub 2015 May 18., [PMID:25987565]
Abstract [show]
P-glycoprotein (P-gp; ABCB1) is an ABC drug pump that protects us from toxic compounds. It is clinically important because it confers multidrug resistance. The homologous halves of P-gp each contain a transmembrane (TM) domain (TMD) with 6 TM segments followed by a nucleotide-binding domain (NBD). The drug- and ATP-binding sites reside at the interface between the TMDs and NBDs, respectively. Each NBD is connected to the TMDs by a transmission interface involving a pair of intracellular loops (ICLs) that form ball-and-socket joints. P-gp is different from CFTR (ABCC7) in that deleting NBD2 causes misprocessing of only P-gp. Therefore, NBD2 might be critical for stabilizing ICLs 2 and 3 that form a tetrahelix bundle at the NBD2 interface. Here we report that the NBD1 and NBD2 transmission interfaces in P-gp are asymmetric. Point mutations to 25 of 60 ICL2/ICL3 residues at the NBD2 transmission interface severely reduced P-gp assembly while changes to the equivalent residues in ICL1/ICL4 at the NBD1 interface had little effect. The hydrophobic nature at the transmission interfaces was also different. Mutation of Phe-1086 or Tyr-1087 to arginine at the NBD2 socket blocked activity or assembly while the equivalent mutations at the NBD1 socket had only modest effects. The results suggest that the NBD transmission interfaces are asymmetric. In contrast to the ICL2/3-NBD2 interface, the ICL1/4-NBD1 transmission interface is more hydrophilic and insensitive to mutations. Therefore the ICL2/3-NBD2 transmission interface forms a precise hydrophobic connection that acts as a linchpin for assembly and trafficking of P-gp.
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No. Sentence Comment
286 Both mutants L443(NBD1)/S909C(IH4) and A266C(IH2)/ F1086C(NBD2) could be cross-linked with copper phenanthroline but only the L443C/S909C mutant was active.
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ABCB1 p.Leu443Cys 25987565:286:126
status: NEW