ABCC7 p.Gln1280Cys
| Predicted by SNAP2: | A: N (61%), C: N (61%), D: N (61%), E: N (66%), F: N (57%), G: D (53%), H: N (97%), I: N (53%), K: N (78%), L: D (53%), M: N (66%), N: N (72%), P: D (53%), R: N (57%), S: N (66%), T: N (66%), V: N (57%), W: D (59%), Y: N (66%), |
| Predicted by PROVEAN: | A: N, C: D, D: N, E: N, F: N, G: D, H: N, I: D, K: N, L: N, M: N, N: N, P: N, R: N, S: N, T: N, V: N, W: D, Y: N, |
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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
72 Cross-linking of C276/Q1280C and C276/K1284C confirms interaction of CL2 and NBD2.
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ABCC7 p.Gln1280Cys 18305154:72:22
status: NEW101 (Right, Bottom two) Controls showing the lack of effects of cross-linking of the Cys-less construct and individually expressed and mixed single cysteine constructs (labeled 276C ϩ Q1280C) under the same conditions (see also SI Fig. 8).
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ABCC7 p.Gln1280Cys 18305154:101:186
status: NEW103 (Left) There is greater propensity for disulfide bond formation at the CL2/NBD2 interface (276C/Q1280C) than at the CL4/NBD1 interface.
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ABCC7 p.Gln1280Cys 18305154:103:96
status: NEW113 In fact, the Q1280C and K1284C substitutions in NBD2 of Cys-less CFTR containing the native Cys-276 residue in CL2 (illustrated in Fig. 3A, Top Right) enabled cross-linking by all MTS reagents tested.
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ABCC7 p.Gln1280Cys 18305154:113:13
status: NEW130 (Middle) Cys-less CFTR with 276C/Q1280C (n ϭ 3).
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ABCC7 p.Gln1280Cys 18305154:130:33
status: NEW[hide] Restoration of domain folding and interdomain asse... FASEB J. 2010 Aug;24(8):3103-12. Epub 2010 Mar 16. He L, Aleksandrov LA, Cui L, Jensen TJ, Nesbitt KL, Riordan JR
Restoration of domain folding and interdomain assembly by second-site suppressors of the DeltaF508 mutation in CFTR.
FASEB J. 2010 Aug;24(8):3103-12. Epub 2010 Mar 16., [PMID:20233947]
Abstract [show]
Deletion of PHE508 (DeltaF508) from the first nucleotide-binding domain (NBD1) of CFTR, which causes most cystic fibrosis, disrupts the folding and assembly of the protein. Although the folding pathways and yield of isolated NBD1 are altered, its global structure is not, and details of the changes in the rest of the protein remain unclear. To gain further insight into how the whole mutant protein is altered, we have determined the influence of known second-site suppressor mutations in NBD1 on the conformation of this domain and key interfaces between domains. We found that the suppressors restored maturation of only those processing mutations located in NBD1, but not in other domains, including those in the C-terminal cytoplasmic loop of the second membrane-spanning domain, which forms an interface with the NBD1 surface. Nevertheless, the suppressors promoted the formation of this interface and others in the absence of F508. The suppressors restored maturation in a DeltaF508 construct from which NBD2 was absent but to a lesser extent than in the full-length, indicating that DeltaF508 disrupts interactions involving NBD2, as well as other domains. Rescue of DeltaF508-CFTR by suppressors required the biosynthesis of the entire full-length protein in continuity, as it did not occur when N- and C-terminal "halves" were coexpressed. Simultaneous with these interdomain perturbations, DeltaF508 resulted in suppressor reversed alterations in accessibility of residues both in the F508-containing NBD1 surface loop and in the Q loop within the domain core. Thus, in the context of the full-length protein, DeltaF508 mutation causes detectable changes in NBD1 conformation, as well as interdomain interactions.
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No. Sentence Comment
122 As shown in Fig. 3A, when ⌬F508 was introduced into Cys-less CFTR containing pairs 276C/Q1280C or 276C/K1284C at the NBD2/CL2 interface, no mature CFTR was detected, and neither M3M nor M8M (200 M) caused cross-linking of the immature band.
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ABCC7 p.Gln1280Cys 20233947:122:95
status: NEW125 As shown in Fig. 3A, ⌬F508-CFTR maturation was restored as was the cross-linking of the Cys pairs 276C/Q1280C and 276C/ K1284C at NBD2/CL2, as indicated by the slower migration rate of the mature bands in SDS-PAGE in samples treated with M3M and M8M (18).
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ABCC7 p.Gln1280Cys 20233947:125:110
status: NEW[hide] Regulatory insertion removal restores maturation, ... J Mol Biol. 2010 Aug 13;401(2):194-210. Epub 2010 Jun 16. Aleksandrov AA, Kota P, Aleksandrov LA, He L, Jensen T, Cui L, Gentzsch M, Dokholyan NV, Riordan JR
Regulatory insertion removal restores maturation, stability and function of DeltaF508 CFTR.
J Mol Biol. 2010 Aug 13;401(2):194-210. Epub 2010 Jun 16., 2010-08-13 [PMID:20561529]
Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR) epithelial anion channel is a large multidomain membrane protein that matures inefficiently during biosynthesis. Its assembly is further perturbed by the deletion of F508 from the first nucleotide-binding domain (NBD1) responsible for most cystic fibrosis. The mutant polypeptide is recognized by cellular quality control systems and is proteolyzed. CFTR NBD1 contains a 32-residue segment termed the regulatory insertion (RI) not present in other ATP-binding cassette transporters. We report here that RI deletion enabled F508 CFTR to mature and traffic to the cell surface where it mediated regulated anion efflux and exhibited robust single chloride channel activity. Long-term pulse-chase experiments showed that the mature DeltaRI/DeltaF508 had a T(1/2) of approximately 14 h in cells, similar to the wild type. RI deletion restored ATP occlusion by NBD1 of DeltaF508 CFTR and had a strong thermostabilizing influence on the channel with gating up to at least 40 degrees C. None of these effects of RI removal were achieved by deletion of only portions of RI. Discrete molecular dynamics simulations of NBD1 indicated that RI might indirectly influence the interaction of NBD1 with the rest of the protein by attenuating the coupling of the F508-containing loop with the F1-like ATP-binding core subdomain so that RI removal overcame the perturbations caused by F508 deletion. Restriction of RI to a particular conformational state may ameliorate the impact of the disease-causing mutation.
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No. Sentence Comment
154 HEK 293 cells were transiently transfected with Cys-less ΔF508 CFTR with Cys pairs introduced at the CL2-NBD2 (C276-Q1280C) or CL4-NBD1 (V510C-G1069C) interfaces in the absence or presence of ΔRI.
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ABCC7 p.Gln1280Cys 20561529:154:122
status: NEW[hide] Mistargeted MRPdeltaF728 mutant is rescued by intr... FEBS Lett. 2004 Dec 3;578(1-2):145-51. Buyse F, Vandenbranden M, Ruysschaert JM
Mistargeted MRPdeltaF728 mutant is rescued by intracellular GSH.
FEBS Lett. 2004 Dec 3;578(1-2):145-51., [PMID:15581632]
Abstract [show]
The most common cystic fibrosis-causing mutation is the deletion of the widely conserved phenylalanine 508 (DeltaF508) of CFTR. The mutant is unable to fold correctly and to transit to the plasma membrane. MRP1 belongs to the same subfamily of ABC proteins as CFTR and confers resistance to a wide range of chemotherapeutic drugs. By analogy, phenylalanine 728 was deleted in MRP1. Our results shown that MRPDeltaF728 is correctly targeted to the plasma membrane, actively transports doxorubicin (DOX) and vincristine (VCR) and shares a structure identical to MRP1. Intracellular GSH depletion however results in a mistargeted mutant that is retained into the cytoplasm, while in the same conditions wild-type MRP1 is correctly routed to the plasma membrane. The GSH-protein complex could adopt a stable conformation protected against proteolytic degradation and correctly targeted to the plasma membrane.
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No. Sentence Comment
154 HEK 293 cells were transiently transfected with Cys-less ΔF508 CFTR with Cys pairs introduced at the CL2-NBD2 (C276-Q1280C) or CL4- NBD1 (V510C-G1069C) interfaces in the absence or presence of ΔRI.
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ABCC7 p.Gln1280Cys 15581632:154:122
status: NEW[hide] Correctors of DeltaF508 CFTR restore global confor... FASEB J. 2013 Feb;27(2):536-45. doi: 10.1096/fj.12-216119. Epub 2012 Oct 26. He L, Kota P, Aleksandrov AA, Cui L, Jensen T, Dokholyan NV, Riordan JR
Correctors of DeltaF508 CFTR restore global conformational maturation without thermally stabilizing the mutant protein.
FASEB J. 2013 Feb;27(2):536-45. doi: 10.1096/fj.12-216119. Epub 2012 Oct 26., [PMID:23104983]
Abstract [show]
Most cystic fibrosis is caused by the deletion of a single amino acid (F508) from CFTR and the resulting misfolding and destabilization of the protein. Compounds identified by high-throughput screening to improve DeltaF508 CFTR maturation have already entered clinical trials, and it is important to understand their mechanisms of action to further improve their efficacy. Here, we showed that several of these compounds, including the investigational drug VX-809, caused a much greater increase (5- to 10-fold) in maturation at 27 than at 37 degrees C (<2-fold), and the mature product remained short-lived (T(1/2) approximately 4.5 h) and thermally unstable, even though its overall conformational state was similar to wild type, as judged by resistance to proteolysis and interdomain cross-linking. Consistent with its inability to restore thermodynamic stability, VX-809 stimulated maturation 2-5-fold beyond that caused by several different stabilizing modifications of NBD1 and the NBD1/CL4 interface. The compound also promoted maturation of several disease-associated processing mutants on the CL4 side of this interface. Although these effects may reflect an interaction of VX-809 with this interface, an interpretation supported by computational docking, it also rescued maturation of mutants in other cytoplasmic loops, either by allosteric effects or via additional sites of action. In addition to revealing the capabilities and some of the limitations of this important investigational drug, these findings clearly demonstrate that DeltaF508 CFTR can be completely assembled and evade cellular quality control systems, while remaining thermodynamically unstable. He, L., Kota, P., Aleksandrov, A. A., Cui, L., Jensen, T., Dokholyan, N. V., Riordan, J. R. Correctors of DeltaF508 CFTR restore global conformational maturation without thermally stabilizing the mutant protein.
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No. Sentence Comment
126 HEK293 cells were transiently transfected with Cys-less CFTR or Cys-less èc;F508 CFTR with Cys pairs 276C/Q1280C or V510C/G1069C, introduced at NBD2/CL2 and NBD1/CL4 interfaces, respectively (13).
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ABCC7 p.Gln1280Cys 23104983:126:110
status: NEW