ABCMdb

ABCC7 p.Ser1248Cys

Predicted by SNAP2:	A: N (66%), C: D (66%), D: D (95%), E: D (95%), F: D (91%), G: D (85%), H: D (91%), I: D (91%), K: D (95%), L: D (91%), M: D (91%), N: D (91%), P: D (95%), Q: D (91%), R: D (95%), T: D (85%), V: D (91%), W: D (95%), Y: D (91%),
Predicted by PROVEAN:	A: N, C: N, D: D, E: N, F: D, G: N, H: D, I: D, K: N, L: D, M: D, N: D, P: D, Q: N, R: D, T: N, V: N, W: D, Y: D,

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[hide] Normal gating of CFTR requires ATP binding to both... Proc Natl Acad Sci U S A. 2005 Jan 11;102(2):455-60. Epub 2004 Dec 27. Berger AL, Ikuma M, Welsh MJ
Normal gating of CFTR requires ATP binding to both nucleotide-binding domains and hydrolysis at the second nucleotide-binding domain.
Proc Natl Acad Sci U S A. 2005 Jan 11;102(2):455-60. Epub 2004 Dec 27., 2005-01-11 [PMID:15623556]

Abstract [show]
ATP interacts with the two nucleotide-binding domains (NBDs) of CFTR to control gating. However, it is unclear whether gating involves ATP binding alone, or also involves hydrolysis at each NBD. We introduced phenylalanine residues into nonconserved positions of each NBD Walker A motif to sterically prevent ATP binding. These mutations blocked [alpha-(32)P]8-N(3)-ATP labeling of the mutated NBD and reduced channel opening rate without changing burst duration. Introducing cysteine residues at these positions and modifying with N-ethylmaleimide produced the same gating behavior. These results indicate that normal gating requires ATP binding to both NBDs, but ATP interaction with one NBD is sufficient to support some activity. We also studied mutations of the conserved Walker A lysine residues (K464A and K1250A) that prevent hydrolysis. By combining substitutions that block ATP binding with Walker A lysine mutations, we could differentiate the role of ATP binding vs. hydrolysis at each NBD. The K1250A mutation prolonged burst duration; however, blocking ATP binding prevented the long bursts. These data indicate that ATP binding to NBD2 allowed channel opening and that closing was delayed in the absence of hydrolysis. The corresponding NBD1 mutations showed relatively little effect of preventing ATP hydrolysis but a large inhibition of blocking ATP binding. These data suggest that ATP binding to NBD1 is required for normal activity but that hydrolysis has little effect. Our results suggest that both NBDs contribute to channel gating, NBD1 binds ATP but supports little hydrolysis, and ATP binding and hydrolysis at NBD2 are key for normal gating.

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111 To directly compare these data with earlier results, we studied S1248C channels (22) and found that NEM altered gating in a similar way (Fig. 2 B and C). Login to comment
113 In addition, correspondence of the S1248F and NEM-modified S1248C data indicate that the gating effects of the S1248F substitution were not due to misfolding that might have occurred during channel biosynthesis. Login to comment
114 For WT and NEM-modified S1248C CFTR, increasing the ATP concentration from 1 mM to 10 mM produced only a small increase in current that was not affected by NEM modification (data not shown), indicating that the effects of NEM represented a block in ATP binding rather than a decreased affinity that could be overcome by increasing ATP concentration. Login to comment
128 (B) Recording from a membrane patch containing a small number of S1248C channels before and after treatment with 200 ␮M NEM. Login to comment
140 The finding that channels unable to bind nucleotide at NBD2 (S1248F and NEM-modified S1248C) had a normal burst duration suggested that the prolonged burst duration of K1250A (16-18, 20, 21) arose when ATP bound NBD2 but then did not undergo hydrolysis. Login to comment
141 To test this hypothesis, we combined the K1250A mutation with S1248C. Login to comment
221 Our results argue against this explanation because blocking NBD2 ATP binding (with either the S1248F mutation or NEM-modification of S1248C) did not prolong burst duration. Login to comment
233 However, it seems surprising that blocking ATP binding to NBD2 (with the S1248F mutation or the NEM-modified S1248C mutation) did not change the rate of channel closure compared to WT. Login to comment

[hide] Control of the CFTR channel's gates. Biochem Soc Trans. 2005 Nov;33(Pt 5):1003-7. Vergani P, Basso C, Mense M, Nairn AC, Gadsby DC
Control of the CFTR channel's gates.
Biochem Soc Trans. 2005 Nov;33(Pt 5):1003-7., [PMID:16246032]

Abstract [show]
Unique among ABC (ATP-binding cassette) protein family members, CFTR (cystic fibrosis transmembrane conductance regulator), also termed ABCC7, encoded by the gene mutated in cystic fibrosis patients, functions as an ion channel. Opening and closing of its anion-selective pore are linked to ATP binding and hydrolysis at CFTR's two NBDs (nucleotide-binding domains), NBD1 and NBD2. Isolated NBDs of prokaryotic ABC proteins form homodimers upon binding ATP, but separate after hydrolysis of the ATP. By combining mutagenesis with single-channel recording and nucleotide photolabelling on intact CFTR molecules, we relate opening and closing of the channel gates to ATP-mediated events in the NBDs. In particular, we demonstrate that two CFTR residues, predicted to lie on opposite sides of its anticipated NBD1-NBD2 heterodimer interface, are energetically coupled when the channels open but are independent of each other in closed channels. This directly links ATP-driven tight dimerization of CFTR's cytoplasmic NBDs to opening of the ion channel in the transmembrane domains. Evolutionary conservation of the energetically coupled residues in a manner that preserves their ability to form a hydrogen bond argues that this molecular mechanism, involving dynamic restructuring of the NBD dimer interface, is shared by all members of the ABC protein superfamily.

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52 We first studied the functional consequence of thiol-specific cross-linking in CFTR channels containing cysteine residues introduced in the NBD1 tail (S549C) and in the NBD2 head (S1248C), in a background similar to that used for the biochemical Figure 2 Statistical coupling analysis detects co-evolution between two positions corresponding to CFTR`s Arg555 (putative hydrogen bond donor) and Thr1246 (putative hydrogen bond acceptor) (A) Side-chain distribution at acceptor position in total multiple sequence alignment (histogram on left) and in each of the two subsets of alignments obtained by 'fixing` the side chain at the donor site, either to an Arg (centre), or to a Lys residue (right). Login to comment

[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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66 'NBD2` composite site, with S549C and S1248C In contrast to these results with single introduced cysteines, BMOE (flexible spacer, reactive groups p8 A˚ apart) or BMH (flexible spacer length, 16 A˚ ) application to oocytes coexpressing CFTR half channels (1-633) S549C and (634-1480) 9CS þ S1248C, with both target cysteines in the NBD2 composite catalytic site (Figure 3), yielded a clear crosslinked product (Figure 6, arrows labeled X-link) not seen without crosslinking reagent (lanes 1 and 9). Login to comment
86 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. Login to comment
95 Western blots identify the NH2-terminal half channel (1-633), S549C (left panel; lower arrow), the COOH-terminal half channel (634-1480) 9CS þ S1248C (right panel; core-glycosylated, B85-90-kDa, bands; fully glycosylated, lower arrow), and cross-linked product (both panels; arrows labeled X-link). Login to comment
120 Although no residual current was seen when 200 mM Cu(II)(o-phenanthroline)2 was added during withdrawal of ATP from split CFTR channels containing only one target cysteine, either S549C (Figure 10A and D) or S1248C (Figure 10B and D), in patches containing both half channels (1-633) S549C and (634-1480) 9CS þ S1248C, a substantial persistent current was observed (Figure 10C and D). Login to comment
123 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. Login to comment
135 Nor did we find convincing evidence for 'homodimeric` interactions of NBD1 or of NBD2 (Figures 5-9): for example, we saw no efficient crosslinking between two NH2-terminal half chan- 0.00 0.04 0.08 0.12 0.16 0.20 S549C S1248C S549C/S1248C (1-633) and (634-1480) 9CS+S1248C(1-633) S549C and (634-1480) 9CS (1-633) S549C and (634-1480) 9CS+S1248C DTT ATP+PKA DTT DTTATP+PKA ATP+PKA ATP+PKA ATP+PKA ATP+PKADTT Cu(phen)2 Cu(phen)2 Cu(phen)2 DTT DTT 50 s 50 s 100 pA100 pA 50 s 100 pA Closure in bath solution Closure in Cu(phen)2 I0 I0/Imax Imax BA C D *** 5 5 7 7 9 10 Figure 10 Functional consequence of crosslinking S549C to S1248C. Login to comment
136 (A-C) Currents activated by 5 mM ATP and 300 nM PKA catalytic subunit in thousands of split CFTR channels in inside-out patches excised from oocytes expressing NH2-terminal (1-633), and COOH-terminal (634-1480) 9CS, half channels containing only one target cysteine, S549C (A) or S1248C (B), or both S549C and S1248C (C). Login to comment
139 For S549C/S1248C channels I0/Imax is significantly different (***Pp0.0005, Student`s t-test) after closure in the presence of Cu(II)(o-phenanthroline)2 compared to its absence (bath solution). Login to comment
159 Functional consequences of crosslinking The nucleotide-independent residual current induced by Cu(II)(o-phenanthroline)2 in the split channels comprising (1-633) S549C and (634-1480) 9CS þ S1248C (Figure 10C) provides direct evidence that artificial stabilization of the NBD1-NBD2 heterodimer tends to keep the affected channels open. Login to comment
160 Detailed characterization of that stabilized open state must await further analysis, but preliminary recordings from patches containing few Cu(II)(o-phenanthroline)2-modified channels show that the disulfide bond between S549C and S1248C results in a high channel open probability in the absence of ATP, with a persistent open state repeatedly interrupted by temporary closures. Login to comment
187 Primers for cysteine insertions S434C, S459C, A462C, S549C, S605C, S1248C, D1336C, S1347C, A1374C and V1379C are given in Table I. Login to comment
199 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. Login to comment

[hide] Covalent modification of the nucleotide binding do... J Biol Chem. 1998 Nov 27;273(48):31873-9. Cotten JF, Welsh MJ
Covalent modification of the nucleotide binding domains of cystic fibrosis transmembrane conductance regulator.
J Biol Chem. 1998 Nov 27;273(48):31873-9., 1998-11-27 [PMID:9822656]

Abstract [show]
The cytosolic nucleotide binding domains of cystic fibrosis transmembrane conductance regulator (NBD1 and NBD2) mediate ATP-dependent opening and closing of the Cl- channel pore. To learn more about NBD structure and function, we introduced a cysteine residue into the Walker A motif or the LSGGQ motif of each NBD and examined modification by N-ethylmaleimide (NEM). Covalent modification of either Walker A motif partially inhibited cystic fibrosis transmembrane conductance regulator channel activity, decreasing the open state probability by prolonging the long closed duration. An increase in cytosolic ATP concentration slowed the rate of modification. The data suggest that both NBDs interact with ATP to influence channel opening and that inhibition by NEM modification was in part due to decreased ATP binding. When cysteine was placed in the NBD2 Walker A motif, it was modified more rapidly than when it was placed in NBD1, suggesting that the NBDs are not structurally or functionally identical. Modification of a cysteine inserted in the LSGGQ motif of either NBD1 or NBD2 also inhibited channel activity. The rate of modification was comparable with that of a thiol in free solution, suggesting that the LSGGQ motif resides in a surface-exposed position in both NBDs.

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No. Sentence Comment
37 1 The abbreviations used are: CFTR, cystic fibrosis transmembrane conductance regulator; NEM, N-ethylmaleimide; PKA, catalytic subunit of cAMP-dependent protein kinase; ABC, ATP-binding cassette; NBD, nucleotide binding domain; NBD1-Cys, A462C/C832A; NBD2-Cys, C832A/S1248C; TB, mean burst duration; g, single channel conductance; ␶cs, slow, long closed time interval; ␶o, open time interval; ␶, time constant for rate of NEM modification; Iϱ, percentage of current remaining following complete NEM modification; TES, N-tris[hydroxymethyl]methyl-2-aminoethanesulfonic acid. Login to comment
73 NEM inhibits NBD1-Cys (CFTR-A462C/C832A) and NBD2-Cys(CFTR-S1248C/C832A) channel activity in an ATP-dependent manner. A, CFTR-C832A; B, CFTR-NBD1-Cys; C, CFTR-NBD2-Cys. Login to comment

[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
259 Evidence that S549 (NBD1 tail) does indeed closely approach Walker A residue S1248 (NBD2 head) across the dimer interface in open CFTR channels comes from the demonstration that a Cu2+ -phenanthroline- induced disulfide bond between cysteine pair S549C and S1248C keeps the channels in prolonged open burst states long after removal of ATP (Mense et al., 2006). Login to comment