ABCMdb

ABCC7 p.Arg1358Ala

CF databases:	c.4074A>T , p.Arg1358Ser (CFTR1) ? , Asymptomatic subject
Predicted by SNAP2:	A: D (91%), C: D (91%), D: D (95%), E: D (95%), F: D (95%), G: D (95%), H: D (85%), I: D (91%), K: D (85%), L: D (91%), M: D (91%), N: D (91%), P: D (95%), Q: D (91%), S: D (91%), T: D (91%), V: D (91%), W: D (95%), Y: D (95%),
Predicted by PROVEAN:	A: D, 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, S: D, T: D, V: D, W: D, Y: D,

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Publications
[hide] Involvement of F1296 and N1303 of CFTR in induced-... J Gen Physiol. 2010 Oct;136(4):407-23. Szollosi A, Vergani P, Csanady L
Involvement of F1296 and N1303 of CFTR in induced-fit conformational change in response to ATP binding at NBD2.
J Gen Physiol. 2010 Oct;136(4):407-23., [PMID:20876359]

Abstract [show]
The chloride ion channel cystic fibrosis transmembrane conductance regulator (CFTR) displays a typical adenosine trisphosphate (ATP)-binding cassette (ABC) protein architecture comprising two transmembrane domains, two intracellular nucleotide-binding domains (NBDs), and a unique intracellular regulatory domain. Once phosphorylated in the regulatory domain, CFTR channels can open and close when supplied with cytosolic ATP. Despite the general agreement that formation of a head-to-tail NBD dimer drives the opening of the chloride ion pore, little is known about how ATP binding to individual NBDs promotes subsequent formation of this stable dimer. Structural studies on isolated NBDs suggest that ATP binding induces an intra-domain conformational change termed "induced fit," which is required for subsequent dimerization. We investigated the allosteric interaction between three residues within NBD2 of CFTR, F1296, N1303, and R1358, because statistical coupling analysis suggests coevolution of these positions, and because in crystal structures of ABC domains, interactions between these positions appear to be modulated by ATP binding. We expressed wild-type as well as F1296S, N1303Q, and R1358A mutant CFTR in Xenopus oocytes and studied these channels using macroscopic inside-out patch recordings. Thermodynamic mutant cycles were built on several kinetic parameters that characterize individual steps in the gating cycle, such as apparent affinities for ATP, open probabilities in the absence of ATP, open probabilities in saturating ATP in a mutant background (K1250R), which precludes ATP hydrolysis, as well as the rates of nonhydrolytic closure. Our results suggest state-dependent changes in coupling between two of the three positions (1296 and 1303) and are consistent with a model that assumes a toggle switch-like interaction pattern during the intra-NBD2 induced fit in response to ATP binding. Stabilizing interactions of F1296 and N1303 present before ATP binding are replaced by a single F1296-N1303 contact in ATP-bound states, with similar interaction partner toggling occurring during the much rarer ATP-independent spontaneous openings.

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No. Sentence Comment
18 We expressed wild-type as well as F1296S, N1303Q, and R1358A mutant CFTR in Xenopus oocytes and studied these channels using macroscopic inside-out patch recordings. Login to comment
112 Fig. S7 shows example macroscopic current traces to illustrate the apparent affinities of R1358A and R1358A/N1303Q for ATP. Login to comment
129 To perturb site 3, we chose mutation R1358A, which is likely to prevent a possible hydrogen-bonding interaction between the R1358 and N1303 side chains. Login to comment
186 (A) Representative traces of R1358A and R1358A/N1303Q currents illustrating segments in 0 mM ATP and bracketing segments in 2 mM ATP. Dotted lines show zero current level. Login to comment
187 (B) Estimation of Po;max for WT (black), R1358A (red), N1303Q (blue), and R1358A/N1303Q (green) by stationary noise analysis. Login to comment
188 Estimated Po;max was 0.62 ± 0.05 for R1358A and 0.36 ± 0.04 for R1358A/N1303Q. Login to comment
191 (E) ATP-dependent current fractions (II0)/(ImaxI0) plotted as a function of [ATP] for WT (black), R1358A (red), N1303Q (blue), and R1358A/N1303Q (green). Login to comment
196 Combining Po;bas/Po;max, obtained from current segments in 0 mM and bracketing periods in 2 mM ATP with Po;max estimated for the 2-mM ATP segments using stationary noise analysis (Fig. 7 B), provided Po;bas estimates (Fig. 7 C) that were higher in both R1358A and R1358A/N1303Q compared with WT or N1303Q. Login to comment
199 We also investigated a possible change in coupling between sites 2 and 3 upon ATP binding by studying [ATP] dependence of macroscopic currents (sample current traces for R1358A and R1358A/N1303Q are shown in Fig. S7, A and B). Login to comment
200 Fitting the [ATP] dose-response curve of the ATP-sensitive current fractions (Fig. 7 E) yielded a slightly increased KPo value for R1358A/N1303Q (inset), but for the calculated KrCO values (Fig. 7 F), a similar trend was apparent even for R1358A. Login to comment
214 Truncation of the site-3 arginine side chain promotes spontaneous, ATP-independent opening regardless of the side chain at site 2 To determine the functional importance of site 3 within our triad of target residues (Fig. 1), we investigated functional coupling between sites 2 (position 1303) and 3 (position 1358) by comparing the effects of removal of the R1358 side chain (R1358A) in either a WT or an N1303Q background. Login to comment
215 Interestingly, after prephosphorylation by 300 nM PKA, both R1358A and R1358A/N1303Q result from formation of a stabilizing interaction in state B (or breaking of a destabilizing interaction present in state A). Login to comment
281 The observed facilitation of spontaneous channel openings by the R1358A mutation (Fig. 7 C) therefore likely reflects loss of a stabilizing interaction in the C1 state between R1358 and a residue other than N1303. Login to comment

[hide] Allosteric coupling between the intracellular coup... PLoS One. 2013 Sep 18;8(9):e74347. doi: 10.1371/journal.pone.0074347. eCollection 2013. Dawson JE, Farber PJ, Forman-Kay JD
Allosteric coupling between the intracellular coupling helix 4 and regulatory sites of the first nucleotide-binding domain of CFTR.
PLoS One. 2013 Sep 18;8(9):e74347. doi: 10.1371/journal.pone.0074347. eCollection 2013., [PMID:24058550]

Abstract [show]
Cystic fibrosis is caused by mutations in CFTR (cystic fibrosis transmembrane conductance regulator), leading to folding and processing defects and to chloride channel gating misfunction. CFTR is regulated by ATP binding to its cytoplasmic nucleotide-binding domains, NBD1 and NBD2, and by phosphorylation of the NBD1 regulatory insert (RI) and the regulatory extension (RE)/R region. These regulatory effects are transmitted to the rest of the channel via NBD interactions with intracellular domain coupling helices (CL), particularly CL4. Using a sensitive method for detecting inter-residue correlations between chemical shift changes in NMR spectra, an allosteric network was revealed within NBD1, with a construct lacking RI. The CL4-binding site couples to the RI-deletion site and the C-terminal residues of NBD1 that precede the R region in full-length CFTR. Titration of CL4 peptide into NBD1 perturbs the conformational ensemble in these sites with similar titration patterns observed in F508del, the major CF-causing mutant, and in suppressor mutants F494N, V510D and Q637R NBD1, as well as in a CL4-NBD1 fusion construct. Reciprocally, the C-terminal mutation, Q637R, perturbs dynamics in these three sites. This allosteric network suggests a mechanism synthesizing diverse regulatory NBD1 interactions and provides biophysical evidence for the allosteric coupling required for CFTR function.

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Sentences [show]
No. Sentence Comment
265 ATP-independent channel opening has been enhanced by Cys, Ser, and Pro mutations of K978 in the ICDs [15] and F1296S/N1303Q and R1358A in NBD2 [60]. Login to comment