ABCC7 p.Trp401Tyr
| ClinVar: |
c.1202G>A
,
p.Trp401*
D
, Pathogenic
c.1203G>A , p.Trp401* D , Pathogenic |
| CF databases: |
c.1202G>A or c.1203G>A
,
p.Trp401*
D
, CF-causing
|
| Predicted by SNAP2: | A: D (75%), C: D (71%), D: D (85%), E: D (85%), F: N (53%), G: D (85%), H: D (80%), I: D (75%), K: D (85%), L: D (59%), M: D (75%), N: D (85%), P: D (91%), Q: D (80%), R: D (80%), S: D (85%), T: D (85%), V: D (63%), Y: N (57%), |
| 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, R: D, S: D, T: D, V: D, Y: D, |
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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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No. Sentence Comment
124 Similar macroscopic experiments were conducted with W401I and W401Y mutations.
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ABCC7 p.Trp401Tyr 20421370:124:62
status: NEW125 The shorter time constant was not significantly affected by either of the mutations (Fig. 3 D), whereas the second time constant (Fig. 3 E) was shortened by nonaromatic substitutions of W401 (W401I) but increased by the conservative W401Y mutation.
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ABCC7 p.Trp401Tyr 20421370:125:233
status: NEW[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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No. Sentence Comment
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).
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ABCC7 p.Trp401Tyr 20861014:4:190
status: NEW34 Here, guided by accumulated structural and functional understanding of NBDs, we first identified W401Y,W401F mutations that strengthened ligand binding in the NBD1 head subdomain.
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ABCC7 p.Trp401Tyr 20861014:34:97
status: NEW36 The presence of the Asp-551 residue, which disfavors NBD dimer formation, however, raised the question of whether ATP molecule potentiates W401Y,W401F/G551D channels by binding at the NBD interface (i.e. site 1) or interacting only with the monomeric NBD1.
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ABCC7 p.Trp401Tyr 20861014:36:139
status: NEW100 Interestingly, we found that phenylalanine (W401F) appeared better than tyrosine (W401Y), which was in turn superior to tryptophan, in stabilizing the lock-open state, suggesting that W401Y and W401F mutations might enhance nucleotide-NBD1 interactions.
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ABCC7 p.Trp401Tyr 20861014:100:82
status: NEWX
ABCC7 p.Trp401Tyr 20861014:100:184
status: NEW103 We then incorporated W401Y or W401F mutation into G551D channels, intending to help G551D-NBD1 to bind nucleotide more tightly.
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ABCC7 p.Trp401Tyr 20861014:103:21
status: NEW106 It can be seen that W401F, which yielded a more stable lock-open state in the WT background (Fig. 1D), was also more effective than W401Y in conferring ATP-dependent activation of G551D channels.
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ABCC7 p.Trp401Tyr 20861014:106:132
status: NEW110 First, the response to ATP seen in W401Y,W401F/G551D channels could be due to an altered ligand-NBD1 interaction as proposed, or alternatively, is a result of a restored ability for ATP to gate CFTR through binding to site 2.
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ABCC7 p.Trp401Tyr 20861014:110:35
status: NEW112 Moreover, we also found that mutating the Trp-401-equivalent residue in NBD2 (Y1219G), which greatly decreases the NBD2 ATP affinity (19), had little effect on ATP-mediated activation of W401Y,W401F/ G551D channels (supplemental Fig. S4).
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ABCC7 p.Trp401Tyr 20861014:112:187
status: NEW113 Second, as PPi fails to lock open G551D-containing channels (data not shown), presumably due to the negatively charged Asp-551 side chain in site 2, it is desirable to have another parameter in support of the conjecture that W401Y,W401F mutations do tighten nucleotide binding in G551D-NBD1.
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ABCC7 p.Trp401Tyr 20861014:113:225
status: NEW114 One possible approach is to fit the current relaxation traces of W401Y,W401F/G551D channels upon removal of the nucleotide (Fig. 2A, inset) as the resulting time constants could potentially reflect the mean nucleotide dwell time in NBD1 of these channels (see also under "Discussion").
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ABCC7 p.Trp401Tyr 20861014:114:65
status: NEW118 Does the ATP or PATP molecule that potentiates W401Y,W401F/G551D channels bind in the head subdomain of the monomeric NBD1 or NBD1-NBD2 dimer interface (i.e. site 1)?
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ABCC7 p.Trp401Tyr 20861014:118:47
status: NEW122 These results thus suggest that ATP or PATP resides in site 1 and interacts with both NBD1 head and NBD2 tail subdomains to support gating of W401Y,W401F/G551D channels.
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ABCC7 p.Trp401Tyr 20861014:122:142
status: NEW123 The idea that both the head of NBD1 and the tail of NBD2 are involved in ATP-dependent activation of W401Y,W401F/ G551D channels implicates that optimizing the interactions of ATP with the NBD2 tail subdomain may also improve the func- FIGURE 2.
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ABCC7 p.Trp401Tyr 20861014:123:101
status: NEW124 W401Y and W401F mutations confer ATP-dependent activa- tionofG551Dchannels.A,applicationofATPorPATPsignificantlyincreased currents of W401Y,W401F/G551D channels.
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ABCC7 p.Trp401Tyr 20861014:124:0
status: NEWX
ABCC7 p.Trp401Tyr 20861014:124:134
status: NEW127 C, effects of W401Y,W401F mutations on the relaxation time constants upon removal of ATP or PATP as shown in panel A. Note that for G551D-CFTR (Trp-401), no current decay was seen because the effect of ATP was negligible.
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ABCC7 p.Trp401Tyr 20861014:127:14
status: NEW145 Single-channel kinetics of G551D channels with W401Y,W401F or H1348G mutations.
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ABCC7 p.Trp401Tyr 20861014:145:47
status: NEW156 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.
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ABCC7 p.Trp401Tyr 20861014:156:191
status: NEW165 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).
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ABCC7 p.Trp401Tyr 20861014:165:134
status: NEW176 Here, we reported that ATP binding in CFTR site 1 is not optimized and can be improved by converting Trp-401 to Tyr/Phe and His-1348 back to Gly, the ABC protein consensus.
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ABCC7 p.Trp401Tyr 20861014:176:101
status: NEW191 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.
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ABCC7 p.Trp401Tyr 20861014:191:140
status: NEW195 The results shown in Fig. 2C did suggest that W401Y,W401F mutations enhance nucleotide binding in G551D site 1, like in the case of WT site 1.
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ABCC7 p.Trp401Tyr 20861014:195:46
status: NEW206 Unfortunately, we cannot use this approach to estimate the Po for other compound mutants, such as W401Y/G551D, as the Po values for these channels are likely too small for noise analysis to be accurate (see supplemental text).
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ABCC7 p.Trp401Tyr 20861014:206:98
status: NEW