ABCC7 p.Leu408Cys
| Predicted by SNAP2: | A: D (53%), C: N (61%), D: D (66%), E: D (59%), F: N (87%), G: D (66%), H: D (53%), I: N (93%), K: D (63%), M: N (78%), N: D (59%), P: D (63%), Q: N (53%), R: D (63%), S: D (53%), T: N (61%), V: N (87%), W: N (53%), Y: N (57%), |
| Predicted by PROVEAN: | A: N, C: N, D: N, E: N, F: N, G: N, H: N, I: N, K: N, M: N, N: N, P: N, Q: N, R: N, S: N, T: N, V: N, W: N, Y: N, |
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[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
116 Based on the latter model, we attempted to detect cross-linking of the residue pairs V171C/E407C and V171C/L408C in the whole protein, but did not observe any indication of cross-linking of the mature band (supplemental Fig. S2A).
X
ABCC7 p.Leu408Cys 18658148:116:107
status: NEW119 To test this hypothesis, membrane vesicles were prepared from BHK cells overexpressing Cys-less CFTR containing V171C and L408C.
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ABCC7 p.Leu408Cys 18658148:119:122
status: NEW127 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).
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ABCC7 p.Leu408Cys 18658148:127:114
status: NEW135 Membrane vesicles prepared from BHK cells overexpressing Cys-less CFTR containing the Cys pair V171C and L408C were treated with 20 M MTS cross-linker M8M before limited trypsin digestion with concentrations indicated in the figures.
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ABCC7 p.Leu408Cys 18658148:135:105
status: NEW172 Turning from the effects of cross-linking CL3 to those of its counterpart, CL1 in the N-terminal MSD, its cross-linking to NBD1 i.e. V171C/L408C had essentially no influence on gating (Fig. 6C).
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ABCC7 p.Leu408Cys 18658148:172:139
status: NEW203 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.
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ABCC7 p.Leu408Cys 18658148:203:94
status: NEW244 We have verified the latter conformation by introducing Cys pairs in CL1 and different residues in RI (V171C/ E407C, V171C/L408C) and showing that these pairs can be cross-linked by M8M (Fig. 3B).
X
ABCC7 p.Leu408Cys 18658148:244:123
status: NEW