ABCC7 p.Cys590Ser
| Predicted by SNAP2: | A: D (53%), D: D (91%), E: D (91%), F: D (91%), G: D (80%), H: D (85%), I: D (85%), K: D (75%), L: D (80%), M: D (85%), N: D (85%), P: D (91%), Q: D (85%), R: D (91%), S: D (71%), T: D (75%), V: D (75%), W: D (91%), Y: D (85%), |
| Predicted by PROVEAN: | A: D, D: D, E: D, F: D, G: D, H: D, I: N, K: D, L: D, M: D, N: D, P: D, Q: D, R: D, S: D, T: D, V: N, W: D, Y: D, |
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[hide] The DeltaF508 mutation disrupts packing of the tra... J Biol Chem. 2004 Sep 17;279(38):39620-7. Epub 2004 Jul 21. Chen EY, Bartlett MC, Loo TW, Clarke DM
The DeltaF508 mutation disrupts packing of the transmembrane segments of the cystic fibrosis transmembrane conductance regulator.
J Biol Chem. 2004 Sep 17;279(38):39620-7. Epub 2004 Jul 21., 2004-09-17 [PMID:15272010]
Abstract [show]
The most common mutation in cystic fibrosis (deletion of Phe-508 in the first nucleotide binding domain (DeltaF508)) in the cystic fibrosis transmembrane conductance regulator (CFTR) causes retention of the mutant protein in the endoplasmic reticulum. We previously showed that the DeltaF508 mutation causes the CFTR protein to be retained in the endoplasmic reticulum in an inactive and structurally altered state. Proper packing of the transmembrane (TM) segments is critical for function because the TM segments form the chloride channel. Here we tested whether the DeltaF508 mutation altered packing of the TM segments by disulfide cross-linking analysis between TM6 and TM12 in wild-type and DeltaF508 CFTRs. These TM segments were selected because TM6 appears to line the chloride channel, and cross-linking between these TM segments has been observed in the CFTR sister protein, the multidrug resistance P-glycoprotein. We first mapped potential contact points in wild-type CFTR by cysteine mutagenesis and thiol cross-linking analysis. Disulfide cross-linking was detected in CFTR mutants M348C(TM6)/T1142C(TM12), T351C(TM6)/T1142C(TM12), and W356C(TM6)/W1145C(TM12) in a wild-type background. The disulfide cross-linking occurs intramolecularly and was reducible by dithiothreitol. Introduction of the DeltaF508 mutation into these cysteine mutants, however, abolished cross-linking. The results suggest that the DeltaF508 mutation alters interactions between the TM domains. Therefore, a potential target to correct folding defects in the DeltaF508 mutant of CFTR is to identify compounds that promote correct folding of the TM domains.
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No. Sentence Comment
57 The construction of Cys-less CFTR (C76S/C126S/C225S/C276S/C343S/C491S/C524S/C590S/C592S/C657S/C832S/C866S/C1344S/C1355S/C1395S/C1400S/C1410S/C1458S) was performed using the following cDNA fragments.
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ABCC7 p.Cys590Ser 15272010:57:76
status: NEW58 Point mutations C76/126S were generated in sequence in the PstI (bp 1) 3 XbaI (bp 573) fragment; point mutations C225S/C276S/C343S were generated in sequence in the XbaI (bp 573) 3 KpnI (bp 1370) fragment; point mutations C491S/C524S/C590S/C592S/C657S were generated in sequence in the KpnI (bp 1370) 3 ApaI (bp 2333) fragment; point mutations C832S/C866S were generated in sequence in the ApaI (bp 2333) 3 EcoRI (bp 3643) fragment; point mutations C1344S/C1355S/ C1395S/C1400S/C1410S/C1458S were generated in sequence in the EcoRI (bp 3643) 3 XhoI (bp 4560) fragment, the five insert fragments were then ligated and inserted into the PstI and XhoI sites of plasmid vector pMT21.
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ABCC7 p.Cys590Ser 15272010:58:234
status: NEW[hide] In vivo phosphorylation of CFTR promotes formation... EMBO J. 2006 Oct 18;25(20):4728-39. Epub 2006 Oct 12. Mense M, Vergani P, White DM, Altberg G, Nairn AC, Gadsby DC
In vivo phosphorylation of CFTR promotes formation of a nucleotide-binding domain heterodimer.
EMBO J. 2006 Oct 18;25(20):4728-39. Epub 2006 Oct 12., 2006-10-18 [PMID:17036051]
Abstract [show]
The human ATP-binding cassette (ABC) protein CFTR (cystic fibrosis transmembrane conductance regulator) is a chloride channel, whose dysfunction causes cystic fibrosis. To gain structural insight into the dynamic interaction between CFTR's nucleotide-binding domains (NBDs) proposed to underlie channel gating, we introduced target cysteines into the NBDs, expressed the channels in Xenopus oocytes, and used in vivo sulfhydryl-specific crosslinking to directly examine the cysteines' proximity. We tested five cysteine pairs, each comprising one introduced cysteine in the NH(2)-terminal NBD1 and another in the COOH-terminal NBD2. Identification of crosslinked product was facilitated by co-expression of NH(2)-terminal and COOH-terminal CFTR half channels each containing one NBD. The COOH-terminal half channel lacked all native cysteines. None of CFTR's 18 native cysteines was found essential for wild type-like, phosphorylation- and ATP-dependent, channel gating. The observed crosslinks demonstrate that NBD1 and NBD2 interact in a head-to-tail configuration analogous to that in homodimeric crystal structures of nucleotide-bound prokaryotic NBDs. CFTR phosphorylation by PKA strongly promoted both crosslinking and opening of the split channels, firmly linking head-to-tail NBD1-NBD2 association to channel opening.
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No. Sentence Comment
25 Functional expression was markedly diminished when C590 and C592 were both mutated to serine, regardless of whether the other 16 cysteines remained (data not shown) or had all been replaced by serines (Figure 2B, 16CS þ C590S/ C592S).
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ABCC7 p.Cys590Ser 17036051:25:225
status: NEW