ABCC7 p.Thr547Cys
| Predicted by SNAP2: | A: D (75%), C: D (75%), D: D (85%), E: D (85%), F: D (85%), G: D (85%), H: D (85%), I: D (85%), K: D (91%), L: D (85%), M: D (80%), N: N (66%), P: D (85%), Q: D (80%), R: D (91%), S: N (57%), V: D (85%), W: D (91%), Y: D (85%), |
| Predicted by PROVEAN: | A: N, C: D, D: N, E: N, F: D, G: N, H: N, I: D, K: N, L: D, M: D, N: N, P: D, Q: N, R: N, S: N, V: D, W: D, Y: D, |
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[hide] Mutations at the signature sequence of CFTR create... J Gen Physiol. 2009 Jan;133(1):69-77. Wang X, Bompadre SG, Li M, Hwang TC
Mutations at the signature sequence of CFTR create a Cd(2+)-gated chloride channel.
J Gen Physiol. 2009 Jan;133(1):69-77., [PMID:19114635]
Abstract [show]
The canonical sequence LSGGQ, also known as the signature sequence, defines the adenosine triphosphate (ATP)-binding cassette transporter superfamily. Crystallographic studies reveal that the signature sequence, together with the Walker A and Walker B motifs, forms the ATP-binding pocket upon dimerization of the two nucleotide-binding domains (NBDs) in a head-to-tail configuration. The importance of the signature sequence is attested by the fact that a glycine to aspartate mutation (i.e., G551D) in cystic fibrosis transmembrane conductance regulator (CFTR) results in a severe phenotype of cystic fibrosis. We previously showed that the G551D mutation completely eliminates ATP-dependent gating of the CFTR chloride channel. Here, we report that micromolar [Cd(2+)] can dramatically increase the activity of G551D-CFTR in the absence of ATP. This effect of Cd(2+) is not seen in wild-type channels or in G551A. Pretreatment of G551D-CFTR with the cysteine modification reagent 2-aminoethyl methane thiosulfonate hydrobromide protects the channel from Cd(2+) activation, suggesting an involvement of endogenous cysteine residue(s) in mediating this effect of Cd(2+). The mutants G551C, L548C, and S549C, all in the signature sequence of CFTR's NBD1, show robust response to Cd(2+). On the other hand, negligible effects of Cd(2+) were seen with T547C, Q552C, and R553C, indicating that a specific region of the signature sequence is involved in transmitting the signal of Cd(2+) binding to the gate. Collectively, these results suggest that the effect of Cd(2+) is mediated by a metal bridge formation between yet to be identified cysteine residue(s) and the engineered aspartate or cysteine in the signature sequence. We propose that the signature sequence serves as a switch that transduces the signal of ligand binding to the channel gate.
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No. Sentence Comment
23 On the other hand, negligible effects of Cd2+ were seen with T547C, Q552C, and R553C, indicating that a specific region of the signature sequence is involved in transmitting the signal of Cd2+ binding to the gate.
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ABCC7 p.Thr547Cys 19114635:23:61
status: NEW104 In contrast, 10 μM Cd2+ shows negligible effect on the T547C mutant (Fig. 6 B).
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ABCC7 p.Thr547Cys 19114635:104:61
status: NEW106 It appears that when cysteine is engineered outside the signature sequence (i.e., T547C and R553C) or at the C-terminal end of the signature sequence (i.e., Q552C), ATP remains a much better ligand than Cd2+ (e.g., Fig. 6 B).
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ABCC7 p.Thr547Cys 19114635:106:82
status: NEW158 (B) Representative T547C current recording in the presence of 1 mM ATP or 10 μM Cd2+ .
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ABCC7 p.Thr547Cys 19114635:158:19
status: NEW168 Fig. 6 demonstrates that the Cd2+ -dependent gating also involves the signature sequence because engineering cysteine residues framing the signature sequence (i.e., T547C and R553C) did not confer this effect of Cd2+ .
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ABCC7 p.Thr547Cys 19114635:168:165
status: NEW