ABCMdb

ABCC7 p.Trp401Tyr

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Publications

PMID: 20421370 [PubMed] Tsai MF et al: "Stable ATP binding mediated by a partial NBD dimer of the CFTR chloride channel."

No. Sentence Comment

124 Similar macroscopic experiments were conducted with W401I and W401Y mutations. Login to comment
125 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. Login to comment

PMID: 20861014 [PubMed] Tsai MF et al: "Optimization of the degenerated interfacial ATP binding site improves the function of disease-related mutant cystic fibrosis transmembrane conductance regulator (CFTR) channels."

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). Login to comment
34 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. Login to comment
36 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. Login to comment
100 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. Login to comment
103 We then incorporated W401Y or W401F mutation into G551D channels, intending to help G551D-NBD1 to bind nucleotide more tightly. Login to comment
106 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. Login to comment
110 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. Login to comment
112 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). Login to comment
113 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. Login to comment
114 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"). Login to comment
118 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)? Login to comment
122 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. Login to comment
123 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. Login to comment
124 W401Y and W401F mutations confer ATP-dependent activa- tionofG551Dchannels.A,applicationofATPorPATPsignificantlyincreased currents of W401Y,W401F/G551D channels. Login to comment
127 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. 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
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
176 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. 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
195 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. Login to comment
206 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). Login to comment