ABCB1 p.Trp803Cys
| Predicted by SNAP2: | A: D (85%), C: D (75%), D: D (91%), E: D (91%), F: N (53%), G: D (85%), H: D (91%), I: D (85%), K: D (91%), L: D (85%), M: D (85%), N: D (91%), P: D (95%), Q: D (91%), R: D (91%), S: D (91%), T: D (85%), V: D (80%), Y: N (57%), |
| Predicted by PROVEAN: | A: D, 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, S: D, T: D, V: D, Y: D, |
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[hide] Locking intracellular helices 2 and 3 together ina... J Biol Chem. 2014 Jan 3;289(1):229-36. doi: 10.1074/jbc.M113.527804. Epub 2013 Nov 25. Loo TW, Clarke DM
Locking intracellular helices 2 and 3 together inactivates human P-glycoprotein.
J Biol Chem. 2014 Jan 3;289(1):229-36. doi: 10.1074/jbc.M113.527804. Epub 2013 Nov 25., [PMID:24275649]
Abstract [show]
The P-glycoprotein (P-gp) drug pump (ABCB1) has two transmembrane domains and two nucleotide-binding domains (NBDs). Coupling of the drug-binding sites in the transmembrane domains to the NBDs occurs through interaction of the intracellular helices (IHs) with residues in the NBDs (IH1/IH4/NBD1 and IH2/IH3/NBD2). We showed previously that cross-linking of cysteines in IH3 and IH1 with a short cross-linker mimicked drug binding as it activated P-gp ATPase activity. Here we show that residue A259C(IH2) could be directly cross-linked to W803C(IH3). Cross-linking was inhibited by the presence of ATP and adenosine 5'-(beta,gamma-imino)triphosphate but not by ADP. Cross-linking of mutant A259C/W803C inhibited its verapamil-stimulated ATPase activity mutant, but activity was restored after addition of dithiothreitol. Because these residues are close to the ball-and-socket joint A266C(IH2)/Phe(1086)(NBD2), we mutated the adjacent Tyr(1087)(NBD2) close to IH3. Mutants Y1087A and Y1087L, but not Y1087F, were misprocessed, and all inhibited ATPase activity. Mutation of hydrophobic residues (F793A, L797A, L814A, and L818A) flanking IH3 also inhibited maturation. The results suggest that these residues, together with Trp(803) and Phe(804), form a large hydrophobic pocket. The results show that there is an important hydrophobic network at the IH2/IH3/NBD2 transmission interface that is critical for folding and activity of P-gp.
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No. Sentence Comment
9 Cross-linking of mutant A259C/W803C inhibited its verapamil-stimulated ATPase activity mutant, but activity was restored after addition of dithiothreitol.
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ABCB1 p.Trp803Cys 24275649:9:30
status: NEW123 It was found that the yield of all the F804C double mutants was significantly lower than the W803C double cysteine mutants (data not shown).
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ABCB1 p.Trp803Cys 24275649:123:93
status: NEW125 Therefore, the W803C double mutants were tested for cross-linking with cupric chloride oxidant.
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ABCB1 p.Trp803Cys 24275649:125:15
status: NEW128 Cross-linked product was observed in all mutants (data not shown), with the greatest amount present in mutant A259C/W803C (about 75%, Fig. 4A).
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ABCB1 p.Trp803Cys 24275649:128:116
status: NEW129 We then tested whether cross-linking of mutant A259C/ W803C could be inhibited by nucleotides.
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ABCB1 p.Trp803Cys 24275649:129:54
status: NEW137 To test the effect of cross-linking on activity, histidine-tagged mutant A259C/W803C was expressed in HEK 293 cells in the presence of cyclosporine A and P-gp isolated by nickel-chelate chromatography.
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ABCB1 p.Trp803Cys 24275649:137:79
status: NEW156 Cross-linking of A259C (IH2) to W803C (IH3) inhibits activity.
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ABCB1 p.Trp803Cys 24275649:156:32
status: NEW157 A, membranes prepared from cells expressing histidine-tagged mutant A259C/ W803C were treated without (afa;) or with (af9;) 0.5 mM CuCl2 for 10 min at 0 &#b0;C in the absence (None) or presence of 5 mM ATP, adenosine 5b18;-(beta,ॹ- imino)triphosphate(AMP.PNP),orADP.Thereactionswerestoppedbyaddition of SDS sample buffer containing no reducing agent, and samples were then subjected to immunoblot analysis.
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ABCB1 p.Trp803Cys 24275649:157:75
status: NEW158 The positions of the cross-linked (X-link) and mature (170-kDa) P-gps are indicated. B, the amount of cross-linked protein relative to total P-gp (170-kDa plus 150-kDa P-gp) was quantitated from three different transfections af9; S.D. C, the histidine-tagged mutant A259C/ W803C was isolated by nickel-chelate chromatography, reconstituted with lipid, and treated without (afa;) or with (af9;) 0.2 mM CuCl2 for 10 min at 20 &#b0;C. The reaction was stopped with 1 mM EDTA.
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ABCB1 p.Trp803Cys 24275649:158:276
status: NEW199 In support of these predictions, we found that clamping IH2 to IH3 by oxidative cross-linking of mutant A259C/W803C or mutations to Tyr1087 inhibited activity.
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ABCB1 p.Trp803Cys 24275649:199:110
status: NEW209 Cross-linking of A259C (blue) in ICL2 to W803C (green) in ICL3 inhibited activity.
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ABCB1 p.Trp803Cys 24275649:209:41
status: NEW[hide] Identification of the distance between the homolog... J Biol Chem. 2014 Mar 21;289(12):8484-92. doi: 10.1074/jbc.M114.552075. Epub 2014 Feb 12. Loo TW, Clarke DM
Identification of the distance between the homologous halves of P-glycoprotein that triggers the high/low ATPase activity switch.
J Biol Chem. 2014 Mar 21;289(12):8484-92. doi: 10.1074/jbc.M114.552075. Epub 2014 Feb 12., [PMID:24523403]
Abstract [show]
P-glycoprotein (P-gp, ABCB1) is an ATP-binding cassette drug pump that protects us from toxic compounds and confers multidrug resistance. Each homologous half contains a transmembrane domain with six transmembrane segments followed by a nucleotide-binding domain (NBD). The drug- and ATP-binding sites reside at the interface between the transmembrane domain and NBDs, respectively. Drug binding activates ATPase activity by an unknown mechanism. There is no high resolution structure of human P-gp, but homology models based on the crystal structures of bacterial, mouse, and Caenorhabditis elegans ATP-binding cassette drug pumps yield both open (NBDs apart) and closed (NBDs together) conformations. Molecular dynamics simulations predict that the NBDs can be separated over a range of distances (over 20 A). To determine the distance that show high or low ATPase activity, we cross-linked reporter cysteines L175C (N-half) and N820C (C-half) with cross-linkers of various lengths that separated the halves between 6 and 30 A (alpha-carbons). We observed that ATPase activity increased over 10-fold when the cysteines were cross-linked at distances between 6 and 19 A, although cross-linking at distances greater than 20 A yielded basal levels of activity. The results suggest that the ATPase activation switch appears to be turned on or off when L175C/N820 are clamped at distances less than or greater than 20 A, respectively. We predict that the high/low ATPase activity switch may occur at a distance where the NBDs are predicted in molecular dynamic simulations to undergo pronounced twisting as they approach each other (Wise, J. G. (2012) Biochemistry 51, 5125-5141).
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No. Sentence Comment
81 P-gp ATPase Activity Is Highly Activated When Residues L175C and N820C Are Cross-linked in Close Proximity-In a previous study, we found that cross-linking the two homologous halves through formation of a direct disulfide bond between A259C in intracellular helix 2 (IH2) and W803C in IH3 inhibited activity (49).
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ABCB1 p.Trp803Cys 24523403:81:276
status: NEW82 It is possible that the A259C/W803C cross-link inhibited activity because these residues are located within IH segments that are predicted to form critical TMD/ NBD contact points.
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ABCB1 p.Trp803Cys 24523403:82:30
status: NEW207 The P-gp molecular dynamics simulation study (64) may also provide an explanation for the inhibition of ATPase activity previously observed when cysteines A295C (in IH2 that connects TM segments 4 and 5) and W803C (in IH3 that connects TM segments 9 and 10) were directly cross-linked (49).
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ABCB1 p.Trp803Cys 24523403:207:208
status: NEW209 It is possible that direct cross-linking of A295C (in IH2 that connects TM segments 4 and 5) to W803C in IH3 would interfere with straightening of TM segments 4 and 5 to trap P-gp in an inactive conformation.
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ABCB1 p.Trp803Cys 24523403:209:96
status: NEW[hide] Translocation mechanism of P-glycoprotein and conf... Biochim Biophys Acta. 2014 Nov;1838(11):2882-98. doi: 10.1016/j.bbamem.2014.07.018. Epub 2014 Jul 25. Prajapati R, Sangamwar AT
Translocation mechanism of P-glycoprotein and conformational changes occurring at drug-binding site: Insights from multi-targeted molecular dynamics.
Biochim Biophys Acta. 2014 Nov;1838(11):2882-98. doi: 10.1016/j.bbamem.2014.07.018. Epub 2014 Jul 25., [PMID:25068895]
Abstract [show]
P-glycoprotein (P-gp) is well known for multidrug resistance in drug therapy. Its over-expression results into the increased efflux of therapeutic agents rendering them inefficacious. A clear understanding of P-gp efflux mechanism and substrate/inhibitor interactions during the course of efflux cycle will be crucial for designing effective P-gp inhibitors, and therapeutic agents that are non-substrate to P-gp. In the present work, we have modeled P-gp in three different catalytic states. These models were utilized for elucidation of P-gp translocation mechanism using multi-targeted molecular dynamics (MTMD). The gradual changes occurring in P-gp structure from inward open to outward open conformation were sampled out. A detailed investigation of conformational changes occurring in trans-membrane domains (TMDs) during the course of catalytic cycle was carried out. Movements of each TM helices in response to pronounced twisting and translatory motion of NBDs were measured quantitatively. The role of intracellular coupling helices (ICHs) during the structural transition of P-gp was studied, and observed as vital links for structural transition. A close observation of displacements and conformational changes in the residues lining drug-binding pocket was also carried out. Further, we have analyzed the molecular interactions of P-gp substrates/inhibitors during the P-gp translocation to find out how stable binding interactions of a compound at drug-binding site(s) in open conformation, becomes highly destabilized in closed conformation. The study revealed striking differences between the molecular interactions of substrate and inhibitor; inhibitors showed a tendency to maintain stable binding interactions during the catalytic transition cycle.
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No. Sentence Comment
462 In this experiment Loo and Clarke cross-linked A259C (ICH2) and W803C (ICH3) that resulted in loss of P-gp ATPase activity.
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ABCB1 p.Trp803Cys 25068895:462:64
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
220 Cysteines were placed in the N-terminal regions of IH1 and IH4 as this was the strategy employed to identify disulfide cross-linking between IH2 (A259C) and IH3 (W803C).
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ABCB1 p.Trp803Cys 25987565:220:162
status: NEW[hide] Mapping the Binding Site of the Inhibitor Tariquid... J Biol Chem. 2015 Dec 4;290(49):29389-401. doi: 10.1074/jbc.M115.695171. Epub 2015 Oct 26. Loo TW, Clarke DM
Mapping the Binding Site of the Inhibitor Tariquidar That Stabilizes the First Transmembrane Domain of P-glycoprotein.
J Biol Chem. 2015 Dec 4;290(49):29389-401. doi: 10.1074/jbc.M115.695171. Epub 2015 Oct 26., [PMID:26507655]
Abstract [show]
ABC (ATP-binding cassette) transporters are clinically important because drug pumps like P-glycoprotein (P-gp, ABCB1) confer multidrug resistance and mutant ABC proteins are responsible for many protein-folding diseases such as cystic fibrosis. Identification of the tariquidar-binding site has been the subject of intensive molecular modeling studies because it is the most potent inhibitor and corrector of P-gp. Tariquidar is a unique P-gp inhibitor because it locks the pump in a conformation that blocks drug efflux but activates ATPase activity. In silico docking studies have identified several potential tariquidar-binding sites. Here, we show through cross-linking studies that tariquidar most likely binds to sites within the transmembrane (TM) segments located in one wing or at the interface between the two wings (12 TM segments form 2 divergent wings). We then introduced arginine residues at all positions in the 12 TM segments (223 mutants) of P-gp. The rationale was that a charged residue in the drug-binding pocket would disrupt hydrophobic interaction with tariquidar and inhibit its ability to rescue processing mutants or stimulate ATPase activity. Arginines introduced at 30 positions significantly inhibited tariquidar rescue of a processing mutant and activation of ATPase activity. The results suggest that tariquidar binds to a site within the drug-binding pocket at the interface between the TM segments of both structural wings. Tariquidar differed from other drug substrates, however, as it stabilized the first TM domain. Stabilization of the first TM domain appears to be a key mechanism for high efficiency rescue of ABC processing mutants that cause disease.
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No. Sentence Comment
65 Membranes were prepared and samples were incubated at 0 &#b0;C (A80C/R741C, A259C/W803C, I299C/F770C, and A266C/ F1086C) or 20 &#b0;C (L175C/N820C, C431/L1176C, and L521C/C1074) for 10 min in the presence or absence of 0.5 Mapping the P-glycoprotein Tariquidar-binding Site 29390 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 290ߦNUMBER 49ߦDECEMBER 4, 2015 at SEMMELWEIS UNIV OF MEDICINE on December 11, mM copper phenanthroline (oxidant to promote disulfide bond formation) in the presence or absence of 0.25 òe;M (for TM segment cysteine mutants) or 1 òe;M tariquidar (for the ICL and NBD cysteine mutants).
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ABCB1 p.Trp803Cys 26507655:65:82
status: NEW112 Accordingly, a Cys-less P-gp (35) was used to generate mutants that contained double cysteines at the interfaces involving TM segments of TMD1 and TMD2 (A80C(TM1)/ R741C(TM7) (29), I299C(TM5)/F770C(TM8) (36), and T333C(TM6/L975C(TM12) (30), or ICL2 and ICL3 (L175C(ICL1)/N820C(ICL3) (23), A259C(ICL2)/W803C- (ICL3) (37), and A266C(ICL2/F1086C(NBD2) (38) (Fig. 1A).
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ABCB1 p.Trp803Cys 26507655:112:301
status: NEW