ABCMdb

ABCB4 p.Ala250Leu

Predicted by SNAP2:	C: D (63%), D: D (66%), E: D (66%), F: D (75%), G: N (57%), H: D (63%), I: D (66%), K: D (71%), L: D (75%), M: D (66%), N: D (59%), P: D (80%), Q: D (63%), R: D (66%), S: N (93%), T: N (53%), V: D (66%), W: D (80%), Y: D (75%),
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: N, T: D, V: D, W: D, Y: D,

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Publications
[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
152 Asymmetry of the P-gp Drug Pump Transmission Interfaces JULY 3, 2015ߦVOLUME 290ߦNUMBER 27 JOURNAL OF BIOLOGICAL CHEMISTRY 16957 at SEMMELWEIS UNIV OF MEDICINE on December 4, twenty-eight mutations in ICL2 (A250L, G251V, V253S, A254L, E256A, L258S, I261S, V264S, F267A, G268V, G269V, L274S, R276A, Y277A) inhibited maturation of P-gp (b0d;15% mature P-gp) while four other ICL2 mutations (A2650L, R262A, T263A, and I265S) partially reduced maturation of P-gp (about 55-60% mature P-gp). Login to comment
160 Rescue of a representative misprocessed mutant by tariquidar is shown on Fig. 3A. No detectable mature P-gp was observed when mutant A250L (ICL2) was expressed in the absence of drug substrate. Login to comment
161 Expression in the presence of tariquidar however, promoted maturation of the A250L mutant to yield mature 170 kDa P-gp as the major product. Login to comment
188 A, representative immunoblot of SDS extracts of whole cells transfected with wild-type P-gp (WT) or mutant A250L. Login to comment