ABCMdb

ABCB1 p.Thr176Cys

Predicted by SNAP2:	A: N (57%), C: D (71%), D: D (91%), E: D (85%), F: D (80%), G: D (80%), H: D (80%), I: D (53%), K: D (85%), L: D (85%), M: D (80%), N: N (61%), P: D (91%), Q: D (75%), R: D (91%), S: N (93%), V: D (75%), W: D (85%), Y: D (85%),
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: N, V: D, W: D, Y: D,

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Publications
[hide] Human P-glycoprotein is active when the two halves... Biochem Biophys Res Commun. 2010 May 7;395(3):436-40. Epub 2010 Apr 13. Loo TW, Bartlett MC, Clarke DM
Human P-glycoprotein is active when the two halves are clamped together in the closed conformation.
Biochem Biophys Res Commun. 2010 May 7;395(3):436-40. Epub 2010 Apr 13., [PMID:20394729]

Abstract [show]
The P-glycoprotein (P-gp, ABCB1) drug pump protects us from toxic compounds and confers multidrug resistance. Each of the two homologous halves of P-gp is composed of a transmembrane domain (TMD) with six 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. Crystal structures show drug pumps in the open and closed conformations, where the drug-binding pocket and NBDs are open or closed at the cytoplasmic side, respectively. Although it has been postulated that drug substrates enter the drug-binding pocket in the open conformation, it is unknown if they can enter in the closed conformation. To determine this, we introduced cysteines into regions of TM3 (residues 175-178) and TM9 (residues 820-822) that extend into the cytoplasm and are 4 A and 20 A apart in the closed and open conformations, respectively. The 12 double cysteine mutants were then cross-linked with a short cross-linker, M1M (4 A) at 0 degrees C to reduce thermal motion in the protein. Only mutant L175C/N820C was cross-linked. Cross-linking was not increased in the presence of ATP or drug substrates. Cross-linking increased its basal ATPase activity about 3-fold. Activity could be increased further by drug substrates such as verapamil and rhodamine B. These results suggest that P-gp in the membrane is in the closed conformation that has a high affinity for drug substrates.

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No. Sentence Comment
97 Verapamil and rhodamine B were selected for study because thiol-reactive derivatives of these drug substrates have been used extensively in cysteine mutagenesis studies to characterize the X-link 170 kDa + + + _ + ++ + + + T176C D177C D178C L175C T176C D177C D178C L175C N820C + D821C + M1M A822C + ++ + T176C D177C D178C L175C B 0.0003 0.001 0.003 0.009 0.027 0.08 0.24 0.73 [M1M] (mM) X-link 170 kDa 0 2.2 C 0 20 40 60 80 100 PercentCross-linked 0.0003 0.001 0.003 0.009 0.027 0.08 0.24 0.73 [M1M] (mM) 0 2.2 A Fig. 2. Login to comment