ABCC7 p.Thr1064Cys
| Predicted by SNAP2: | A: D (71%), C: D (71%), D: D (85%), E: D (85%), F: D (85%), G: D (85%), H: D (85%), I: D (71%), K: D (91%), L: D (80%), M: D (71%), N: D (71%), P: D (85%), Q: D (85%), R: D (91%), S: D (63%), V: D (63%), W: D (91%), 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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[hide] Phenylalanine-508 mediates a cytoplasmic-membrane ... Proc Natl Acad Sci U S A. 2008 Mar 4;105(9):3256-61. Epub 2008 Feb 27. Serohijos AW, Hegedus T, Aleksandrov AA, He L, Cui L, Dokholyan NV, Riordan JR
Phenylalanine-508 mediates a cytoplasmic-membrane domain contact in the CFTR 3D structure crucial to assembly and channel function.
Proc Natl Acad Sci U S A. 2008 Mar 4;105(9):3256-61. Epub 2008 Feb 27., 2008-03-04 [PMID:18305154]
Abstract [show]
Deletion of phenylalanine-508 (Phe-508) from the N-terminal nucleotide-binding domain (NBD1) of the cystic fibrosis transmembrane conductance regulator (CFTR), a member of the ATP-binding cassette (ABC) transporter family, disrupts both its folding and function and causes most cystic fibrosis. Most mutant nascent chains do not pass quality control in the ER, and those that do remain thermally unstable, only partially functional, and are rapidly endocytosed and degraded. Although the lack of the Phe-508 peptide backbone diminishes the NBD1 folding yield, the absence of the aromatic side chain is primarily responsible for defective CFTR assembly and channel gating. However, the site of interdomain contact by the side chain is unknown as is the high-resolution 3D structure of the complete protein. Here we present a 3D structure of CFTR, constructed by molecular modeling and supported biochemically, in which Phe-508 mediates a tertiary interaction between the surface of NBD1 and a cytoplasmic loop (CL4) in the C-terminal membrane-spanning domain (MSD2). This crucial cytoplasmic membrane interface, which is dynamically involved in regulation of channel gating, explains the known sensitivity of CFTR assembly to many disease-associated mutations in CL4 as well as NBD1 and provides a sharply focused target for small molecules to treat CF. In addition to identifying a key intramolecular site to be repaired therapeutically, our findings advance understanding of CFTR structure and function and provide a platform for focused biochemical studies of other features of this unique ABC ion channel.
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No. Sentence Comment
81 In addition to the Phe-508-containing NBD1 surface patch, residues in other regions of the domain also interact with CL4 residues as evidenced by cross-linking of Cys pairs involving amino acids closer to the Q loop (Gln-493), including W496C/T1064C and M498C/L1065C as well as nearer the Walker B motif (Asp-572) such as K564C/G1069C (Fig. 3A and SI Fig. 8).
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ABCC7 p.Thr1064Cys 18305154:81:243
status: NEW97 Phe-508 participates in an apparent aromatic cluster with residues from CL4(seealsoSIFig.10).CL4alsointeractswithotherregionsinNBD1assuggested by cross-linking of residues close to the Q loop (W496C/T1064C and M498C/ L1065C) and a residue near the Walker B motif (K564C/G1069C).
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ABCC7 p.Thr1064Cys 18305154:97:199
status: NEW[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
80 In agreement with what is predicted by our model, we were able to cross-link residue pairs W496C/T1064C, M498C/L1065C (19), which proves that indeed CL4 also interacts with NBD1 through the so called Q-loop (Q493).
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ABCC7 p.Thr1064Cys 18658148:80:97
status: NEW159 Membrane vesicles prepared from HEK cells transiently transfected with Cys pairs introduced at E543C with T966C (CL3) and T1064C (CL4) were pretreated with PKA catalytic subunit in the presence of ATP before incubating with 20 M MTS reagents.
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ABCC7 p.Thr1064Cys 18658148:159:122
status: NEW