ABCB1 p.Phe904Cys
| Predicted by SNAP2: | A: D (59%), C: N (53%), D: D (80%), E: D (75%), G: D (75%), H: D (59%), I: N (93%), K: D (80%), L: D (63%), M: N (61%), N: D (71%), P: D (85%), Q: D (71%), R: D (80%), S: D (71%), T: D (71%), V: N (87%), W: D (59%), Y: D (66%), |
| Predicted by PROVEAN: | A: D, C: D, D: D, E: D, G: D, H: D, I: N, K: D, L: N, M: N, N: D, P: D, Q: D, R: D, S: D, T: D, V: N, W: D, Y: N, |
[switch to compact view]
Comments [show]
None has been submitted yet.
[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.
Comments [show]
None has been submitted yet.
No. Sentence Comment
92 Disulfide Cross-linking Analysis-Cys-less P-gp or the ICL1/ ICL4 double cysteine mutants I160C/F904C or F163C/R905C were transiently expressed in HEK 293 cells at reduced temperature (30 &#b0;C) to promote maturation.
X
ABCB1 p.Phe904Cys 25987565:92:95
status: NEW223 Mutants I160C/F904C and F163/R905C (Fig. 5, A and B) were selected to test the effects of IH1/IH4 cross-linking as the mutants yielded mature P-gp that showed efficient cross-linking in the presence of oxidant (copper phenanthroline) (Fig. 5C).
X
ABCB1 p.Phe904Cys 25987565:223:14
status: NEW226 No cross-linked product was observed when the I160C, F904C, F163C, or R905C mutants were treated with oxidant (data not shown).
X
ABCB1 p.Phe904Cys 25987565:226:53
status: NEW227 To test for the effects of cross-linking on activity, isolated Cys-less P-gp and mutants I160C/F904C and F163/R905C were treated without or with copper phenanthroline for 10 min a 20 &#b0;C.
X
ABCB1 p.Phe904Cys 25987565:227:95
status: NEW239 Mutants I160C/ F904C and F163C/R905C exhibited over 70% of the activity of the Cys-less parent (Fig. 5D).
X
ABCB1 p.Phe904Cys 25987565:239:15
status: NEW240 Cross-linking of mutants I160C/ F904C and F163C/R905C with oxidant however, inhibited more than 90% of the activity.
X
ABCB1 p.Phe904Cys 25987565:240:32
status: NEW