ABCC7 p.Ala1374Cys
| Predicted by SNAP2: | C: D (59%), D: D (85%), E: D (80%), F: D (80%), G: D (66%), H: D (80%), I: D (75%), K: D (85%), L: D (80%), M: D (75%), N: D (71%), P: D (85%), Q: D (75%), R: D (85%), S: N (87%), T: D (66%), V: D (71%), W: D (85%), Y: D (85%), |
| Predicted by PROVEAN: | 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, T: D, V: D, W: D, Y: D, |
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[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
78 Central region, with S605C and A1374C Similar results were obtained with the pair of cysteines introduced at positions 605 and 1374, predicted to lie near the center of the proposed NBD1-NBD2 interface.
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ABCC7 p.Ala1374Cys 17036051:78:31
status: NEW86 Crosslinking was weaker, but still evident, 250 150 100 75 kDa - - - + + - + - + - - - + + - + - + - - - + + - + - + - - - + + - + - + - - - + + - + - + - - - + + - + - + fsk Anti-R-domainAnti-N-terminus BMOE BMH Background S434C S459C A462C S549C S605C - - - + + - + - + - - - + + - + - + - - - + + - + - + - - - + + - + - + - - - + + - + - + S1248C D1336C S1347C A1374C V1379C 250 150 100 75 50 Figure 5 The absence of efficient crosslinking when no, or only one, engineered cysteine is present.
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ABCC7 p.Ala1374Cys 17036051:86:365
status: NEW123 The six include three crosslinks across the NBD1 composite site (between the NBD1 head, containing the Walker motifs, and the NBD2 tail, containing the ABC signature sequence: C462-C1347, C459-C1379, and C434- C1336), one crosslink between central regions of NBD1 and NBD2 (C605-C1374), one crosslink between the NBD1-tail 250 150 100 75 50 kDa fsk BMOE BMH - - + - + + + - + + - +- - - - + -- + + 0ЊC23ЊC - - + - + + + - + + - +- - - - + -- + + 0ЊC23ЊC fsk BMOE BMH X-link CFTR 1-633 X-link CFTR 634-1480 Anti-R-domainAnti-N-terminus 1 2 3 4 5 6 7 8 9 10 11 12 13 14 (1-633) S549C and (634-1480) 9CS+A1374CB 250 150 100 75 kDa - + + - + - - - + fsk BMOE BMH - + + - + - - - + - + + - + - - - + - + + - + - - - + S459C/S1248C S549C/D1336C S549C/V1379C S605C/D1336C 250 150 50 Anti-R-domainAnti-N-terminus - + + - + - - - + S434C/A1374C A Two engineered cysteine control experiments Figure 9 Tests of crosslinking between NBD1 and NBD2 using other combinations of the target cysteines.
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ABCC7 p.Ala1374Cys 17036051:123:853
status: NEW187 Primers for cysteine insertions S434C, S459C, A462C, S549C, S605C, S1248C, D1336C, S1347C, A1374C and V1379C are given in Table I.
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ABCC7 p.Ala1374Cys 17036051:187:91
status: NEW199 For recording macroscopic currents of split CFTR channels in excised patches (Figure 10), oocytes were Table I Forward primers for site-directed mutagenesis PCR C76S 50 -GCCCTTCGGCGATcgTTTTTCTGGAG-30 C276S 50 -CTGTTAAGGCCTACTcCTGGGAAGAAGC-30 C832S 50 -CGAAGAAGACCTTAAGGAGTcCTTTTTTGATGATATGGAGAGC-30 EagI site 50 -GGTAAAATTAAGCACAGcGGccGAATTTCATTCTGTTCTC-30 HA epitope 50 -CGGGCCGCCATGtAcccatAcGACGttccgGAttAcgcaAGGTCGCCTCTGG-30 CFTR 16CS C590A/C592A 50 -GGAGATCTTCGAGAGCgCTGTCgCTAAACTGATGGC-30 CFTR 16CS C590F/C592F 50 -GGAGATCTTCGAGAGCTtTGTCTtTAAACTGATGGC-30 CFTR 16CS C590L/C592L 50 -GGAGATCTTCGAGAGCctTGTCctTAAACTGATGGC-30 CFTR 16CS C590T/C592T 50 -GGAGATCTTCGAGAGCaCTGTCaCTAAACTGATGGC-30 CFTR 16CS C590V/C592V 50 -GGAGATCTTCGAGAGCgtcGTCgtTAAACTGATGGC-30 S434C 50 -CCTCTTCTTCAGTAATTTCTgtCTaCTTGGTACTCCTGTC-30 S459C 50 -GTTGGCGGTTGCTGGATgCACTGGAGCAGGCAAG-3 A462C 50 -GCTGGATCCACTGGGtgcGGCAAGACTTCACTTC-30 L549C 50 -GGTGGAATCACACtatGcGGAGGTCAACGAGCACG-30 S605C 50 -GGATTTTGGTCACaTgTAAAATGGAAC-30 S1248C 50 -CCTCTTGGGAAGAACCGGtTgtGGGAAGAGTAC-30 D1336C 50 -GTTTCCTGGGAAGCTTtgCTTTGTCCTTGTGG-30 L1346C 50 -GGATGGGGGCTCTGTCTgtAGTCATGGCCACAAGC-30 A1374C 50 -GATGAACCAAGCtgTCATTTAGATCC-30 V1379C 50 -GCTCATTTAGATCCgtgcACATACCAAATAATTCG-30 The underlined bases are the codons for the introduced serines, cysteines or other residues; lowercase letters mark base changes from the original sequence, including those for introducing diagnostic restriction endonuclease sites.
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ABCC7 p.Ala1374Cys 17036051:199:1142
status: NEW[hide] Cysteine accessibility probes timing and extent of... J Gen Physiol. 2015 Apr;145(4):261-83. doi: 10.1085/jgp.201411347. Chaves LA, Gadsby DC
Cysteine accessibility probes timing and extent of NBD separation along the dimer interface in gating CFTR channels.
J Gen Physiol. 2015 Apr;145(4):261-83. doi: 10.1085/jgp.201411347., [PMID:25825169]
Abstract [show]
Cystic fibrosis transmembrane conductance regulator (CFTR) channel opening and closing are driven by cycles of adenosine triphosphate (ATP) binding-induced formation and hydrolysis-triggered disruption of a heterodimer of its cytoplasmic nucleotide-binding domains (NBDs). Although both composite sites enclosed within the heterodimer interface contain ATP in an open CFTR channel, ATP hydrolysis in the sole catalytically competent site causes channel closure. Opening of the NBD interface at that site then allows ADP-ATP exchange. But how frequently, and how far, the NBD surfaces separate at the other, inactive composite site remains unclear. We assessed separation at each composite site by monitoring access of nucleotide-sized hydrophilic, thiol-specific methanothiosulfonate (MTS) reagents to interfacial target cysteines introduced into either LSGGQ-like ATP-binding cassette signature sequence (replacing equivalent conserved serines: S549 and S1347). Covalent MTS-dependent modification of either cysteine while channels were kept closed by the absence of ATP impaired subsequent opening upon ATP readdition. Modification while channels were opening and closing in the presence of ATP caused macroscopic CFTR current to decline at the same speed as when the unmodified channels shut upon sudden ATP withdrawal. These results suggest that the target cysteines can be modified only in closed channels; that after modification the attached MTS adduct interferes with ATP-mediated opening; and that modification in the presence of ATP occurs rapidly once channels close, before they can reopen. This interpretation was corroborated by the finding that, for either cysteine target, the addition of the hydrolysis-impairing mutation K1250R (catalytic site Walker A Lys) similarly slowed, by an order of magnitude, channel closing on ATP removal and the speed of modification by MTS reagent in ATP. We conclude that, in every CFTR channel gating cycle, the NBD dimer interface separates simultaneously at both composite sites sufficiently to allow MTS reagents to access both signature-sequence serines. Relatively rapid modification of S1347C channels by larger reagents-MTS-glucose, MTS-biotin, and MTS-rhodamine-demonstrates that, at the noncatalytic composite site, this separation must exceed 8 A.
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No. Sentence Comment
228 A1374C CFTR channels, with the target in the NBD2 D-loop, were also readily modified by MTSET+ applied in the absence (Fig. 11 A) or presence of ATP (Fig. 11 B), in either case leaving a residual current 10-20% of control (Fig. 11 C).
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ABCC7 p.Ala1374Cys 25825169:228:0
status: NEW230 In contrast to the irreversible modification of S605C channels, the MTSET+ adduct could be released from modified A1374C channels by treatment with DTT (Fig. 11, A and B).
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ABCC7 p.Ala1374Cys 25825169:230:114
status: NEW260 In addition, mutant cycle analysis indicates that NBD2 Walker A T1246 hydrogen bonds to NBD1 R555, Figure 11.ߓ Rapid decay of current in A1374C CFTR channels (containing a mid-interface target Cys-in the NBD2 D loop-between the two composite sites) upon ATP washout (w/o) or modification by MTSET+ .
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ABCC7 p.Ala1374Cys 25825169:260:143
status: NEW261 (A and B) A1374C-(C832S-C1458S) CFTR channels were modified by 1 mM MTSET+ (red trace bar) either while closed in the absence of ATP (A), as assessed by subsequent diminished ATP-activated current (3 mM, black bars below records), or in the presence of ATP (B).
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ABCC7 p.Ala1374Cys 25825169:261:10
status: NEW264 (C) Amplitude of residual current (Iresidual %), relative to ATP-activated current before modification, for A1374C channels modified, while closed (left, 0 ATP), by 5 &#b5;M-1 mM MTSET+ (red bar, 9 &#b1; 2%; n = 8 measurements in six patches), or while opening and closing (right, 3 mM ATP), by 10 &#b5;M-1 mM MTSET+ (red bar, 17 &#b1; 6%, n = 3 measurements in two patches).
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ABCC7 p.Ala1374Cys 25825169:264:108
status: NEW280 Analysis of tryptic digests (Aleksandrov et al., 2002, 2008) or of split A1374C, located between the two composite sites, implies separation all along the dimer interface each cycle.
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ABCC7 p.Ala1374Cys 25825169:280:73
status: NEW392 In contrast, the residual current after MTSET+ modification varied with position along the dimer interface, and was &#e07a;4% of control with the adduct at S549C (Fig. 4), 10-20% at A1374C (Fig. 11), &#e07a;20% at S605C (Fig. 10), and &#e07a;40% at S1347C (Fig. 6).
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ABCC7 p.Ala1374Cys 25825169:392:182
status: NEW