ABCMdb

ABCB1 p.Ala266Cys

Predicted by SNAP2:	C: D (66%), D: D (85%), E: D (85%), F: D (85%), G: D (71%), H: D (80%), I: D (80%), K: D (85%), L: D (75%), M: D (53%), N: D (80%), P: D (91%), Q: D (80%), R: D (85%), S: N (66%), T: D (63%), V: D (80%), W: D (91%), Y: D (85%),
Predicted by PROVEAN:	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, W: D, Y: D,

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[hide] Processing mutations disrupt interactions between ... J Biol Chem. 2008 Oct 17;283(42):28190-7. Epub 2008 Aug 16. Loo TW, Bartlett MC, Clarke DM
Processing mutations disrupt interactions between the nucleotide binding and transmembrane domains of P-glycoprotein and the cystic fibrosis transmembrane conductance regulator (CFTR).
J Biol Chem. 2008 Oct 17;283(42):28190-7. Epub 2008 Aug 16., 2008-10-17 [PMID:18708637]

Abstract [show]
P-glycoprotein (P-gp, ABCB1) is an ATP-dependent drug pump. Each of its two homologous halves contains a transmembrane domain (TMD) that has six transmembrane (TM) segments and a nucleotide-binding domain (NBD). Determining how the two halves interact may provide insight into the folding of P-gp as the drug-binding pocket and nucleotide-binding sites are predicted to be at the interface between the two halves. Here, we present evidence for NBD1-TMD2 and NBD2-TMD1 interactions. We also show that TMD-NBD interactions in immature and mature P-gp can be affected by the presence of a processing mutation. We found that the NBD-TMD mutants L443C(NBD1)/S909C(TMD2) and A266C(TMD1)/F1086C(NBD2) could be cross-linked at 0 degrees C with oxidant (copper phenanthroline). Cross-linking was inhibited by vanadate-trapping of nucleotide. The presence of a processing mutation (G268V/L443C(NBD1)/S909C(TMD2); L1260A/A266C(TMD1)/F1086C(NBD2)) resulted in the synthesis of the immature (150 kDa) protein as the major product and the mutants could not be cross-linked with copper phenanthroline. Expression of the processing mutants in the presence of a pharmacological chaperone (cyclosporin A), however, resulted in the expression of mature (170 kDa) protein at the cell surface that could be cross-linked. Similarly, CFTR mutants A274C(TMD1)/L1260C(NBD2) and V510C(NBD1)/A1067C(TMD2) could be cross-linked at 0 degrees C with copper phenanthroline. Introduction of DeltaF508 mutation in these mutants, however, resulted in the synthesis of immature CFTR that could not be cross-linked. These results suggest that establishment of NBD interactions with the opposite TMD is a key step in folding of ABC transporters.

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No. Sentence Comment
107 The locations of positions equivalent to cysteines S473C and R905C in the other half of P-gp (A266C/F1086C) are indicated. Login to comment
151 Because no cross-linking studies have identified cysteines that could be cross-linked at the TMD1-NBD2 interface, we constructed a series of double cysteine mutants between a cysteine in TMD1 (A266C or F267C) and another in NBD2 (R1085C, F1086C, Y1087C or D1088C) that would be equivalent to L443C(NBD1) and S909C(TMD2), respectively, in each half of P-gp. Login to comment
164 A, membranes prepared from cells expressing mutant A266C/F1086C were treated with (ϩ) or without (-) 1 mM copper phenanthroline (CuP) for 15 min at 0 °C. Membranes were also treated with drug substrates or ATP plus vanadate as described in the legend to Fig. 4. Login to comment

[hide] The W232R suppressor mutation promotes maturation ... Biochemistry. 2011 Feb 8;50(5):672-85. Epub 2011 Jan 11. Loo TW, Bartlett MC, Clarke DM
The W232R suppressor mutation promotes maturation of a truncation mutant lacking both nucleotide-binding domains and restores interdomain assembly and activity of P-glycoprotein processing mutants.
Biochemistry. 2011 Feb 8;50(5):672-85. Epub 2011 Jan 11., 2011-02-08 [PMID:21182301]

Abstract [show]
ATP-binding cassette (ABC) proteins contain two nucleotide-binding domains (NBDs) and two transmembrane (TM) domains (TMDs). Interdomain interactions and packing of the TM segments are critical for function, and disruption by genetic mutations contributes to disease. P-glycoprotein (P-gp) is a useful model to identify mechanisms that repair processing defects because numerous arginine suppressor mutations have been identified in the TM segments. Here, we tested the prediction that a mechanism of arginine rescue was to promote intradomain interactions between TM segments and restore interdomain assembly. We found that suppressor W232R(TM4/TMD1) rescued mutants with processing mutations in any domain and restored defective NBD1-NBD2, NBD1-TMD2, and TMD1-TMD2 interactions. W232R also promoted packing of the TM segments because it rescued a truncation mutant lacking both NBDs. The mechanism of W232R rescue likely involved intradomain hydrogen bond interactions with Asn296(TM5) since only N296A abolished rescue by W232R and rescue was only observed when Trp232 was replaced with hydrogen-bonding residues. In TMD2, suppressor T945R(TM11) also promoted packing of the TM segments because it rescued the truncation mutant lacking the NBDs and suppressed formation of alternative topologies. We propose that T945R rescue was mediated by interactions with Glu875(TM10) since T945E/E875R promoted maturation while T945R/E875A did not.

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No. Sentence Comment
164 Accordingly, we introduced the W232R mutation into mutant A266C/F1086C/L1260A. Login to comment
166 Figure 3C shows that the W232R mutation promoted maturation of the mutant A266C/F1086C/L1260A (lane 3) and that only the mature protein was cross-linked (lane 4). Login to comment
188 Membranes were also prepared from cells expressing the L1260A processing mutant ( W232R containing cysteines in TMD1 and NBD2 (A266C/F1086C) (C). Login to comment

[hide] A salt bridge in intracellular loop 2 is essential... Biochemistry. 2013 May 14;52(19):3194-6. doi: 10.1021/bi400425k. Epub 2013 May 3. Loo TW, Clarke DM
A salt bridge in intracellular loop 2 is essential for folding of human p-glycoprotein.
Biochemistry. 2013 May 14;52(19):3194-6. doi: 10.1021/bi400425k. Epub 2013 May 3., [PMID:23634976]

Abstract [show]
There is no high-resolution structure of the human P-glycoprotein (P-gp, ABCB1) drug pump. Homology models based on the crystal structures of mouse and Caenorhabditis elegans P-gps show extensive contacts between intracellular loop 2 (ICL2, in the first transmembrane domain) and the second nucleotide-binding domain. Human P-gp modeled on these P-gp structures yields different ICL2 structures. Only the model based on the C. elegans P-gp structure predicts the presence of a salt bridge. We show that the Glu256-Arg276 salt bridge was critical for P-gp folding.

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13 ICL2 appears to play a key role in coupling NBD1-TMD2 interactions because cysteines introduced into ICL2 (A266C) and NBD2 (F1086C) could be cross-linked and the F1086C change abolished activity.13 While both structures predict that residues 261-267 form an interhelical loop (IH2) (forms the ball portion of the ball-and-socket ICL2-NBD connection), adjacent amino acids were predicted to adopt loop or b1;-helical structures in the mouse or C. elegans structures, respectively. Login to comment

[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
56 IH2 appears to be particularly important because we showed that a cysteine introduced into IH2 (A266C) could be directly cross-linked to a cysteine in NBD2 (F1086C) but the mutant was inactive (21). Login to comment
62 Because mutant A266C/F1086C was inactive, we characterized the Ala-266/ Phe-1086 interface to determine its role in the transport cycle. Login to comment
109 In a previous study, we provided biochemical evidence that Ala-266 was indeed close to Phe-1086 in NBD2 because mutant A266C/F1086C showed robust cross-linking even when treated with oxidant at 0 &#b0;C to slow molecular motion (21). Login to comment
110 The Ala-266/Phe-1086 contact point appeared to be critical for function because mutant A266C/F1086C was inactive (21). Login to comment
117 IH2 Mediates TMD/NBD Coupling in the P-gp Drug Pump 20328 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 288ߦNUMBER 28ߦJULY 12, 2013 To determine whether one or both cysteine mutations in mutant A266C/F1086C inhibited activity, mutants were constructed in a Cys-less background that contained only Cys-266 or Cys-1086. Login to comment
138 A and B, structures of P-gp in the open (A) (17) or closed (B) (24) conformations are shown. C, histidine-tagged Cys-less P-gp or mutants A266C/F1086C, A266C, and F1086C (in Cys-less background) as well as wild-type P-gp and mutant F1086A ( in wild-type background) were isolated, and ATPase activities were measured in the presence of verapamil. Login to comment