ABCMdb

ABCC7 p.Phe1296Ser

Predicted by SNAP2:	A: D (63%), C: D (53%), D: D (91%), E: D (80%), G: D (75%), H: D (75%), I: D (71%), K: D (80%), L: N (66%), M: D (71%), N: D (71%), P: D (80%), Q: D (66%), R: D (75%), S: D (71%), T: D (71%), V: D (66%), W: D (80%), Y: D (75%),
Predicted by PROVEAN:	A: D, C: D, D: D, E: D, G: D, H: D, I: D, K: D, L: D, M: D, N: D, P: D, Q: D, R: D, S: D, T: D, V: D, W: D, Y: N,

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[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
72 In this study, the average durations of stationary segments of record used for estimating Po;max were 40-50 s for the wild-type (WT), F1296S, N1303Q, and F1296S/N1303Q constructs (estimated single-channel cycle times 1.25 s in saturating ATP; Fig. 8 A), but 100-130 s for K1250R, F1296S/K1250R, and N1303Q/K1250R, and 220 s for F1296S/ N1303Q/K1250R (estimated single-channel cycle times 13 s in saturating ATP; Fig. 8 A). Login to comment
109 Figs. S3 and S4 show verification of Po;max estimates in single-channel patches for WT, F1296S, N1303Q, and F1296S/N1303Q (Fig. S3), as well as for K1250R, F1296S/K1250R, N1303Q/K1250R, and F1296S/N1303Q/K1250R (Fig. S4). Login to comment
113 Fig. S8 depicts predicted Po time courses in response to the addition/removal of ATP for WT, F1296S/N1303Q, K1250R, and F1296S/N1303Q/K1250R, calculated using Scheme 2. Login to comment
118 (A-D) Inward chloride currents recorded in patches excised from resting oocytes expressing WT (A), F1296S (B), N1303Q (C), and F1296S/N1303Q (D) CFTR. Login to comment
122 Phosphorylation dependence of channel activity is largely unchanged for F1296S, N1303Q, and F1296S/N1303Q CFTR mutants, whereas the double mutant displays significantly increased ATP-independent activity Similarly to WT CFTR (Fig. 2 A), in patches excised from resting oocytes expressing F1296S (Fig. 2 B), N1303Q (Fig. 2 C), or double mutant F1296S/N1303Q (Fig. 2 D) CFTR channels, we observed very low channel activity upon the application of 2 mM ATP, but robust macroscopic currents could be activated for all three constructs by the subsequent addition of 300 nM PKA to the bath solution. Login to comment
127 This pattern of coevolution between sites 1 and 2 suggests that possible functional interactions between the two residues, perturbed by single mutations F1296S and N1303Q, might be restored in a double mutant F1296S/N1303Q. Login to comment
131 (A) Representative traces of WT, F1296S, N1303Q, and F1296S/N1303Q currents illustrating segments in 0 mM ATP and bracketing segments in 2 mM ATP. Dotted lines show zero current level. Login to comment
132 (B) Estimation of Po;max for WT (black), F1296S (red), N1303Q (blue), and F1296S/N1303Q (green) by stationary noise analysis. Login to comment
138 maximal open probabilities were little changed in all three mutant constructs (0.22 ± 0.02, 0.32 ± 0.04, and 0.33 ± 0.03, respectively, for F1296S, N1303Q, and F1296S/ N1303Q) compared with WT (0.35 ± 0.03) (Fig. 3 B), open probability in zero ATP was 10-fold higher for the F1296S/N1303Q double mutant than for WT or the two single mutants (Fig. 3 C). Login to comment
142 Indeed, although K1250R, F1296S/K1250R, and N1303Q/K1250R ATP removal rapidly abolished currents for both single mutants just as for WT (Fig. 2, A-C), in the case of the double mutant, a constitutive basal activity persisted even after ATP removal (Fig. 2 D, magnified in inset) and did not vanish even over the time course of several minutes. Login to comment
147 (A) Representative traces of K1250R, F1296S/K1250R, N1303Q/K1250R, and F1296S/N1303Q/K1250R currents illustrating segments in 0 mM ATP and bracketing segments in 2 mM ATP. Dotted lines show zero current level (determined for the triple mutant similarly to that in Fig. S2). Login to comment
148 (B) Estimation of Po;max for K1250R (black), F1296S/K1250R (red), N1303Q/K1250R (blue), and F1296S/N1303Q/K1250R (green) by stationary noise analysis. Login to comment
155 constructs showed hardly detectable basal activity, a markedly elevated spontaneous activity was observed for the F1296S/N1303Q/K1250R triple mutant (Fig. 4 A), persisting even minutes after ATP washout. Login to comment
157 Similarly to their hy-drolytic counterparts, Po;bas was 10-fold higher in F1296S/N1303Q/K1250R compared with the other three constructs, and the mutant cycle built on the closed-open equilibrium constant Po;bas/(1Po;bas) yielded a Gint of 2.36 ± 0.58 kT (Fig. 4 D)-again, significantly different from zero (P < 0.01). Login to comment
160 Conventional dwell-time analysis of such records yielded Po;max values similar to those obtained by noise analysis both for the WT, F1296S, N1303Q, and F1296S/ N1303Q constructs (Fig. S3, A and B), and for the Figure 5. Energetic coupling between sites 1 and 2 changes between ATP-bound open and ATP-free closed states, but not between ATP-bound closed and open states. Login to comment
161 (A) Summary of Po;max values for K1250R (black), F1296S/K1250R (red), N1303Q/K1250R (blue), and F1296S/N1303Q/K1250R (green) obtained from the data presented in Fig. 4 B. Login to comment
175 Thus, to test for a possible change in interaction between sites 1 and 2 during ATP-driven reversible opening and closure, we repeated the mutant cycle analysis in the nonhydrolytic K1250R background, comparing Po;max values for K1250R, F1296S/K1250R, N1303Q/ K1250R, and F1296S/N1303Q/K1250R (Fig. 5 A), Figure 6. ATP binding affects energetic coupling between sites 1 and 2 in closed channels. Login to comment
176 (A) [ATP] dependence of macroscopic currents was assayed for WT (top left), F1296S (top right), N1303Q (bottom left), and F1296S/N1303Q (bottom right) channels by exposure to various test [ATP] bracketed by exposures to 2 mM ATP. Login to comment
177 (B) ATP-dependent current fractions (II0)/(ImaxI0) plotted as a function of [ATP] for WT (black), F1296S (red), N1303Q (blue), and F1296S/N1303Q (green). Login to comment
184 Although neither the F1296S nor the N1303Q mutation, when introduced one at a time, affected the time constant of current relaxation of K1250R upon ATP removal (Fig. 5 C, red and blue fit lines and bars), this relaxation time constant (relax) was prolonged by approximately fourfold, to 31 ± 5 s (n = 10), in the triple mutant F1296S/ N1303Q/K1250R (Fig. 5 C, green fit line and bar). Login to comment
205 For instance, in TAP1, the site-1 phenylalanine backbone forms an H bond with the asparagine at site 2 (corresponding to N1303 in CFTR`s NBD2) when ATP is bound to the protein (Procko et al., 2006), whereas in the ADP-bound form Right-shift in ATP dependence of opening rate for F1296S/ N1303Q indicates a change in interaction between sites 1 and 2 upon ATP binding to closed channels To test whether the site-1-site-2 interaction was affected by ATP binding, we evaluated the apparent affinity for ATP to open WT, F1296S, N1303Q, and F1296S/ N1303Q channels. Login to comment
209 Just as for KPo, the resulting KrCO values were slightly lower for F1296S and N1303Q than for WT, but approximately twofold increased for the double mutant F1296S/N1303Q (Fig. 6 C). Login to comment
227 Phenotypic pattern of single and double mutants at site 1 and 2 suggests toggle switch mechanism Using mutants F1296S, N1303Q, and F1296S/N1303Q, we studied the interaction between sites 1 and 2 in the form of mutant cycles built on (a) ATP-independent spontaneous activity (i.e., Po;bas/(1Po;bas)) both in hydrolysis-competent (Fig. 3 D) and (b) in catalytically inactive (Fig. 4 D) background; (c) ATP-dependent maximal activity (i.e., Po;max/(1Po;max)) (Fig. 5 B) and (d) de- activationrateuponATPremoval(Fig.5D)ofcatalytically incompetent constructs; and (e) apparent affinity for ATP to open the channels (Fig. 6 D). Login to comment
228 Interestingly, although four of the above five mutant cycles (Figs. 3 D, 4 D, 5 D, and 6 D) yielded Gint significantly different from zero, in each case this was due to a significantly altered phenotype in the double mutant F1296S/N1303Q, whereas the single mutations F1296S and N1303Q did not measurably affect gating (Figs. 3-6). Login to comment
268 The two rates assumed to be changed by the F1296S/N1303Q double mutation, and by the K1250R mutation, are shown in red and magenta, respectively, belowtheWTrates. Login to comment
269 (B)Tablesummarizingparam- eters Po;bas and KPo predicted by Scheme 2 for WT (using the rates in black in A) and F1296S/ N1303Q (using the two rates in red in A), as well as Po;max and relax for K1250R and F1296S/ N1303Q/K1250R (using the rates printed in magenta for steps C4→O2 and O2→C1). Login to comment
272 in an unchanged Po;max (Fig. 8 B), just as we have observed for F1296S/N1303Q/K1250R (Fig. 5 A). Login to comment
274 Such predicted time courses are summarized in Fig. S8 for WT, F1296S/N1303Q, K1250R, and F1296S/N1303Q/K1250R. Login to comment
275 Thus, this alteration of only two analogous rates in the double mutant F1296S/N1303Q is sufficient to account for all of our data on site-1-site-2 interactions (Figs. 1-6). Login to comment
276 Because S-Q (and A-Q) pairings are also quite frequent alternatives of the most typical F-N pairing at these two sites (Fig. 1 A), it is conceivable that the site-1-site-2 interaction postulated for states C2, C4, O1, and O2 in WT can also be formed to some extent in F1296S/N1303Q. Login to comment
293 The lack of phenotype in the single mutants N1303Q and F1296S can be accounted for by assuming stabilizing F-X and N-Y interactions in states C1 and C3 replaced by a stabilizing F-N interaction in states C2 and C4 (retained in O1 and O2) in WT CFTR (illustrated in the cartoon in Fig. 8 A). Login to comment
295 In contrast, the double mutation F1296S/N1303Q removes two stabilizing interactions (F-X and N-Y) in states C1 and C3, but only one such interaction (F-N) in states C2, C4, O1, and O2. Login to comment
297 Indeed, a 160-fold decrease in rate C2→C1 (Fig. 8 A, red) increases Po;bas by 10-fold (Fig. 8 B) to the level we have observed for F1296S/N1303Q (Fig. 3 C). Login to comment
298 A comparable (100-fold) decrease in rate C4→C3 (Fig. 8 A, red) reproduces the approximately fourfold prolonged relax (Fig. 8 B) we have observed for F1296S/N1303Q/K1250R (Fig. 5 C). Login to comment
300 Moreover, the reduction in rate C4→C3 has no impact on maximal opening rate, and hence on Po;max, consistent with our observation for the double mutant F1296S/N1303Q (Fig. 3 B). 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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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