ABCMdb

ABCC7 p.Leu1261Cys

Predicted by SNAP2:	A: N (93%), C: N (93%), D: D (66%), E: N (61%), F: N (72%), G: N (72%), H: D (53%), I: N (97%), K: N (66%), M: N (87%), N: N (57%), P: N (78%), Q: N (53%), R: N (53%), S: N (87%), T: N (87%), V: N (93%), W: N (53%), Y: N (72%),
Predicted by PROVEAN:	A: N, C: N, D: N, E: N, F: N, G: D, H: D, I: N, K: N, M: N, N: D, P: D, Q: N, R: D, S: N, T: N, V: N, W: D, Y: N,

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Publications
[hide] Multiple membrane-cytoplasmic domain contacts in t... J Biol Chem. 2008 Sep 26;283(39):26383-90. Epub 2008 Jul 25. He L, Aleksandrov AA, Serohijos AW, Hegedus T, Aleksandrov LA, Cui L, Dokholyan NV, Riordan JR
Multiple membrane-cytoplasmic domain contacts in the cystic fibrosis transmembrane conductance regulator (CFTR) mediate regulation of channel gating.
J Biol Chem. 2008 Sep 26;283(39):26383-90. Epub 2008 Jul 25., 2008-09-26 [PMID:18658148]

Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR) is a unique ATP-binding cassette (ABC) ion channel mutated in patients with cystic fibrosis. The most common mutation, deletion of phenylalanine 508 (DeltaF508) and many other disease-associated mutations occur in the nucleotide binding domains (NBD) and the cytoplasmic loops (CL) of the membrane-spanning domains (MSD). A recently constructed computational model of the CFTR three-dimensional structure, supported by experimental data (Serohijos, A. W., Hegedus, T., Aleksandrov, A. A., He, L., Cui, L., Dokholyan, N. V., and Riordan, J. R. (2008) Proc. Natl. Acad. Sci. U. S. A. 105, 3256-3261) revealed that several of these mutations including DeltaF508 disrupted interfaces between these domains. Here we have used cysteine cross-linking experiments to verify all NBD/CL interfaces predicted by the structural model and observed that their cross-linking has a variety of different effects on channel gating. The interdomain contacts comprise aromatic clusters important for stabilization of the interfaces and also involve the Q-loops and X-loops that are in close proximity to the ATP binding sites. Cross-linking of all domain-swapping contacts between NBDs and MSD cytoplasmic loops in opposite halves of the protein rapidly and reversibly arrest single channel gating while those in the same halves have lesser impact. These results reinforce the idea that mediation of regulatory signals between cytoplasmic- and membrane-integrated domains of the CFTR channel apparently relies on an array of precise but highly dynamic interdomain structural joints.

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No. Sentence Comment
106 To confirm these contacts in the case of CL3 and NBD2, we designed several Cys pairs from CL3 (M961C and S962C) and NBD2 (L1260C and L1261C) (Fig. 3A). Login to comment
108 In constructs containing the Cys pairs M961C/L1261C (Fig. 3A), M961C/L1260C, and S962C/ L1261C (supplemental Fig. S1A), MTS reagent treatment produced a slightly, albeit distinguishably, faster moving band, which could be reversed by DTT. Login to comment
111 Cross-linking of Cys pairs between CL3 and NBD2 was also confirmed with co-expression of constructs of Cys-less ⌬NBD2 CFTR containing M961C together with the Cys-less NBD2 fragment containing L1261C in HEK cells (supplemental Fig. S1, B and C). Login to comment
127 No cross-linking was detected when Cys pairs were introduced at L172C/E543C, T966C/D1341C, V171C/L1261C, or M961C/L408C, which are not predicted to be in association in the structural model (supplemental Fig. S3). Login to comment
156 First, cross-linking between residues M961C and L1261C at the CL3/ NBD2 interface changed channel gating behavior substantially but did not arrest it completely (Fig. 6A). Login to comment
203 A, M961C/L1261C at the CL3/NBD2 interface; B, T966C/E543C at the CL3/NBD1 interface; C, V171C/L408C at the CL1/NBD1 interface; D, L171C/D1341C at the CL1/NBD2 interface. Login to comment