ABCMdb

ABCC7 p.His1348Gly

Predicted by SNAP2:	A: N (82%), C: N (61%), D: D (53%), E: N (61%), F: N (66%), G: N (87%), I: N (82%), K: N (72%), L: N (82%), M: N (93%), N: N (82%), P: D (59%), Q: N (82%), R: N (72%), S: N (82%), T: N (72%), V: N (93%), W: N (53%), Y: N (87%),
Predicted by PROVEAN:	A: N, C: N, D: N, E: N, F: N, G: N, I: N, K: N, L: N, M: N, N: N, P: N, Q: N, R: N, S: N, T: N, V: N, W: N, Y: N,

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[hide] Stable ATP binding mediated by a partial NBD dimer... J Gen Physiol. 2010 May;135(5):399-414. Tsai MF, Li M, Hwang TC
Stable ATP binding mediated by a partial NBD dimer of the CFTR chloride channel.
J Gen Physiol. 2010 May;135(5):399-414., [PMID:20421370]

Abstract [show]
Cystic fibrosis transmembrane conductance regulator (CFTR), a member of the adenosine triphosphate (ATP) binding cassette (ABC) superfamily, is an ATP-gated chloride channel. Like other ABC proteins, CFTR encompasses two nucleotide binding domains (NBDs), NBD1 and NBD2, each accommodating an ATP binding site. It is generally accepted that CFTR's opening-closing cycles, each completed within 1 s, are driven by rapid ATP binding and hydrolysis events in NBD2. Here, by recording CFTR currents in real time with a ligand exchange protocol, we demonstrated that during many of these gating cycles, NBD1 is constantly occupied by a stably bound ATP or 8-N(3)-ATP molecule for tens of seconds. We provided evidence that this tightly bound ATP or 8-N(3)-ATP also interacts with residues in the signature sequence of NBD2, a telltale sign for an event occurring at the NBD1-NBD2 interface. The open state of CFTR has been shown to represent a two-ATP-bound NBD dimer. Our results indicate that upon ATP hydrolysis in NBD2, the channel closes into a "partial NBD dimer" state where the NBD interface remains partially closed, preventing ATP dissociation from NBD1 but allowing the release of hydrolytic products and binding of the next ATP to occur in NBD2. Opening and closing of CFTR can then be coupled to the formation and "partial" separation of the NBD dimer. The tightly bound ATP molecule in NBD1 can occasionally dissociate from the partial dimer state, resulting in a nucleotide-free monomeric state of NBDs. Our data, together with other structural/functional studies of CFTR's NBDs, suggest that this process is poorly reversible, implying that the channel in the partial dimer state or monomeric state enters the open state through different pathways. We therefore proposed a gating model for CFTR with two distinct cycles. The structural and functional significance of our results to other ABC proteins is discussed.

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150 Of particular note is the H1348G mutation, which increased the time constant of the second phase (Fig. 5 B), suggesting that this mutation actually prolongs the ATP dwell time in NBD1. Login to comment

[hide] Optimization of the degenerated interfacial ATP bi... J Biol Chem. 2010 Nov 26;285(48):37663-71. Epub 2010 Sep 22. Tsai MF, Jih KY, Shimizu H, Li M, Hwang TC
Optimization of the degenerated interfacial ATP binding site improves the function of disease-related mutant cystic fibrosis transmembrane conductance regulator (CFTR) channels.
J Biol Chem. 2010 Nov 26;285(48):37663-71. Epub 2010 Sep 22., 2010-11-26 [PMID:20861014]

Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel, an ATP binding cassette (ABC) protein whose defects cause the deadly genetic disease cystic fibrosis (CF), encompasses two nucleotide binding domains (NBD1 and NBD2). Recent studies indicate that in the presence of ATP, the two NBDs coalesce into a dimer, trapping an ATP molecule in each of the two interfacial composite ATP binding sites (site 1 and site 2). Experimental evidence also suggests that CFTR gating is mainly controlled by ATP binding and hydrolysis in site 2, whereas site 1, which harbors several non-canonical substitutions in ATP-interacting motifs, is considered degenerated. The CF-associated mutation G551D, by introducing a bulky and negatively charged side chain into site 2, completely abolishes ATP-induced openings of CFTR. Here, we report a strategy to optimize site 1 for ATP binding by converting two amino acid residues to ABC consensus (i.e. H1348G) or more commonly seen residues in other ABC proteins (i.e. W401Y,W401F). Introducing either one or both of these mutations into G551D-CFTR confers ATP responsiveness for this disease-associated mutant channel. We further showed that the same maneuver also improved the function of WT-CFTR and the most common CF-associated DeltaF508 channels, both of which rely on site 2 for gating control. Thus, our results demonstrated that the degenerated site 1 can be rebuilt to complement or support site 2 for CFTR function. Possible approaches for developing CFTR potentiators targeting site 1 will be discussed.

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4 Here, we report a strategy to optimize site 1 for ATP binding by converting two amino acid residues to ABC consensus (i.e. H1348G) or more commonly seen residues in other ABC proteins (i.e. W401Y,W401F). Login to comment
37 Our data suggested that the former is likely the case as the effects of ATP on W401F/G551D channels can be further enhanced by the H1348G mutation in the NBD2 tail subdomain. Login to comment
54 For the W401F/H1348G/G551D channel, the steady-state open probability (Po) in the presence of ATP was estimated by stationary noise analysis of macroscopic currents using the equation ␴2 /i ϭ ͑1 - Po͒I (Eq. 1) where ␴2 is the variance, i is the unitary current amplitude, and I is the amplitude of the steady-state currents. Login to comment
62 This can be readily done for WT and W401F/ H1348G/⌬F508 channels (as seen in Fig. 5). Login to comment
131 We thus converted the His-1348 residue to Gly (H1348G) in the G551D background. Login to comment
133 Moreover, the H1348G mutation further improved the function of W401F/G551D channels so that the application of ATP and PATP increased the basal activity by ϳ25- and ϳ75-fold, respectively (Fig. 3, C and D, and supplemental Fig. S5), further supporting the notion that optimizing ATP binding in site 1 enhances the function of G551D channels. Login to comment
139 A, the S1347G mutation diminished the response of W401F/G551D channels to ATP or PATP. B, incorporating the H1348G mutation into G551D-CFTR conferred responsiveness to ATP. Login to comment
140 C, H1348G enhanced the response of W401F/G551D-CFTR to ATP. Login to comment
145 Single-channel kinetics of G551D channels with W401Y,W401F or H1348G mutations. Login to comment
156 W401F and H1348G Mutations Improve the Function of WT and ⌬F508 Channels-To this point, we have demonstrated that optimizing the interactions of ATP with site 1 components, NBD1 head (W401Y and W401F) and NBD2 tail (H1348G), ameliorates the gating defects of G551D channels, which hold a non-functional site 2. Login to comment
160 Indeed, when W401F and H1348G mutations were engineered into WT channels (Fig. 5A), the mean open time of WT-CFTR was more than quadrupled (Fig. 5C) with the already high Po (ϳ0.4) nearly doubled (ϳ0.78, Fig. 5D). Login to comment
162 In either case, W401F/H1348G mutations did not significantly alter the opening rate (Fig. 5E). Login to comment
165 First, ATP-site 1 interactions of the CFTR channel can be strengthened by introducing mutations in both the head domain of NBD1 (i.e. W401Y,W401F) and the tail domain of NBD2 (i.e. H1348G). Login to comment
180 Effects of W401F/H1348G mutations on WT and ⌬F508 channels. Login to comment
181 A, 30-s single-channel recordings of WT and W401F/H1348G channels exposed to 2.75 mM ATP. Login to comment
182 B, current recordings of ⌬F508 and ⌬F508/W401F/ H1348G channels in the presence of 2.75 mM ATP. Login to comment
191 It is this correlation between the chemical nature of mutations and the stability of the lock-open state that grants us the confidence that W401Y,W401F and H1348G mutations, which prolonged the lock-open duration of WT-CFTR, indeed tighten ATP binding in site 1. Login to comment
199 Unlike WT and ⌬F508 channels, whose Po in the presence or absence of W401F/H1348G mutations (Fig. 5) can be measured with reasonable accuracy, the G551D-containing channels exhibit a Po too low to be derived from single-channel analysis. Login to comment
204 For instance, that the W401F/H1348G/G551D channel has an IATP/IBasal of ϳ25 indicates that the Po for this mutant is ϳ0.1. Login to comment
205 To verify this value, we performed stationary noise analysis for the W401F/ H1348G/G551D mutant, and the resulting Po of 0.09 Ϯ 0.01 (supplemental Fig. S7) did provide some reassurance. Login to comment
209 We have observed that W401F/H1348G mutations in site 1 prolonged the mean open time of WT-CFTR (Fig. 5C). Login to comment
211 Thus, it appears that a stronger ATP binding in site 1, due to the presence of W401F/H1348G mutations, can allosterically tighten the connection between two NBDs around site 2, thereby slowing down channel closure. Login to comment

[hide] The most common cystic fibrosis-associated mutatio... J Physiol. 2011 Jun 1;589(Pt 11):2719-31. Epub 2011 Apr 11. Jih KY, Li M, Hwang TC, Bompadre SG
The most common cystic fibrosis-associated mutation destabilizes the dimeric state of the nucleotide-binding domains of CFTR.
J Physiol. 2011 Jun 1;589(Pt 11):2719-31. Epub 2011 Apr 11., 2011-06-01 [PMID:21486785]

Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel that belongs to the ATP binding cassette (ABC) superfamily. The deletion of the phenylalanine 508 (DeltaF508-CFTR) is the most common mutation among cystic fibrosis (CF) patients. The mutant channels present a severe trafficking defect, and the few channels that reach the plasma membrane are functionally impaired. Interestingly, an ATP analogue, N6-(2-phenylethyl)-2'-deoxy-ATP (P-dATP), can increase the open probability (Po) to approximately 0.7, implying that the gating defect of DeltaF508 may involve the ligand binding domains, such as interfering with the formation or separation of the dimeric states of the nucleotide-binding domains (NBDs). To test this hypothesis, we employed two approaches developed for gauging the stability of the NBD dimeric states using the patch-clamp technique. We measured the locked-open time induced by pyrophosphate (PPi), which reflects the stability of the full NBD dimer state, and the ligand exchange time for ATP/N6-(2-phenylethyl)-ATP (P-ATP), which measures the stability of the partial NBD dimer state wherein the head of NBD1 and the tail of NBD2 remain associated. We found that both the PPi-induced locked-open time and the ATP/P-ATP ligand exchange time of DeltaF508-CFTR channels are dramatically shortened, suggesting that the DeltaF508 mutation destabilizes the full and partial NBD dimer states. We also tested if mutations that have been shown to improve trafficking of DeltaF508-CFTR, namely the solubilizing mutation F494N/Q637R and DeltaRI (deletion of the regulatory insertion), exert any effects on these newly identified functional defects associated with DeltaF508-CFTR. Our results indicate that although these mutations increase the membrane expression and function of DeltaF508-CFTR, they have limited impact on the stability of both full and partial NBD dimeric states for DeltaF508 channels. The structure-function insights gained from this mechanism may provide clues for future drug design.

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No. Sentence Comment
105 Tight binding of nucleotides in NBD1 prolongs the channel locked-open time In a previous report (Tsai et al. 2010a), we demonstrated that the locked-open time of WT-CFTR induced by PPi is prolonged by replacing ATP with the high affinity ATP analogue N6 -phenylethyl-ATP (P-ATP), or by introducing 'gain-of-function` mutations to the ATP-binding site 1 (mutations which increase the Po of CFTR, such as W401F and H1348G) as the locked-open state reflects an NBD dimer with ATP-binding site 1 occupied by ATP and ATP-binding site 2 by PPi (Tsai et al. 2009). Login to comment
106 In Fig. 3, we show that the gain-of-function mutations W401F and H1348G (Fig. 3A) and P-ATP (Fig. 3B) also prolong the locked-open time of F508-CFTR channels. Login to comment
107 Compared to F508-CFTR, the double mutant W401F/ F508-CFTR ( F508/DM)prolongedthelocked-opentimeby~2-fold, and the triple mutant W401F/H1348G/ F508-CFTR ( F508/TM) by ~4-fold. Login to comment
127 C, summary of PPi locked-open times for each construct ( F508/DM: W401F/ F508-CFTR, F508/TM: W401F/H1348G/ F508-CFTR). Login to comment