ABCMdb

ABCC7 p.Ser573Glu

CF databases:	c.1718C>G , p.Ser573Cys (CFTR1) ? , c.1718C>T , p.Ser573Phe (CFTR1) ? ,
Predicted by SNAP2:	A: N (82%), C: D (63%), D: N (72%), E: N (57%), F: D (85%), G: N (72%), H: D (80%), I: D (80%), K: D (59%), L: D (80%), M: D (80%), N: N (72%), P: D (59%), Q: N (61%), R: D (85%), T: N (78%), V: D (75%), W: D (91%), Y: D (85%),
Predicted by PROVEAN:	A: N, C: D, D: N, E: N, F: D, G: N, H: N, I: D, K: N, L: D, M: D, N: N, P: N, Q: N, R: D, T: N, V: D, W: D, Y: D,

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Publications
[hide] On the mechanism of MgATP-dependent gating of CFTR... J Gen Physiol. 2003 Jan;121(1):17-36. Vergani P, Nairn AC, Gadsby DC
On the mechanism of MgATP-dependent gating of CFTR Cl- channels.
J Gen Physiol. 2003 Jan;121(1):17-36., [PMID:12508051]

Abstract [show]
CFTR, the product of the gene mutated in cystic fibrosis, is an ATPase that functions as a Cl(-) channel in which bursts of openings separate relatively long interburst closed times (tauib). Channel gating is controlled by phosphorylation and MgATP, but the underlying molecular mechanisms remain controversial. To investigate them, we expressed CFTR channels in Xenopus oocytes and examined, in excised patches, how gating kinetics of phosphorylated channels were affected by changes in [MgATP], by alterations in the chemical structure of the activating nucleotide, and by mutations expected to impair nucleotide hydrolysis and/or diminish nucleotide binding affinity. The rate of opening to a burst (1/tauib) was a saturable function of [MgATP], but apparent affinity was reduced by mutations in either of CFTR's nucleotide binding domains (NBDs): K464A in NBD1, and K1250A or D1370N in NBD2. Burst duration of neither wild-type nor mutant channels was much influenced by [MgATP]. Poorly hydrolyzable nucleotide analogs, MgAMPPNP, MgAMPPCP, and MgATPgammaS, could open CFTR channels, but only to a maximal rate of opening approximately 20-fold lower than attained by MgATP acting on the same channels. NBD2 catalytic site mutations K1250A, D1370N, and E1371S were found to prolong open bursts. Corresponding NBD1 mutations did not affect timing of burst termination in normal, hydrolytic conditions. However, when hydrolysis at NBD2 was impaired, the NBD1 mutation K464A shortened the prolonged open bursts. In light of recent biochemical and structural data, the results suggest that: nucleotide binding to both NBDs precedes channel opening; at saturating nucleotide concentrations the rate of opening to a burst is influenced by the structure of the phosphate chain of the activating nucleotide; normal, rapid exit from bursts occurs after hydrolysis of the nucleotide at NBD2, without requiring a further nucleotide binding step; if hydrolysis at NBD2 is prevented, exit from bursts occurs through a slower pathway, the rate of which is modulated by the structure of the NBD1 catalytic site and its bound nucleotide. Based on these and other results, we propose a mechanism linking hydrolytic and gating cycles via ATP-driven dimerization of CFTR's NBDs.

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154 The mean closing rate from bursts was not substantially altered by these NBD1 mutations (compare Fig. 5 E and Table I): for D572N, rOC(5 mM MgATP ϩ PKA) ϭ 1.4 Ϯ 0.2 s-1 (n ϭ 9), and rOC(5 mM MgATP) ϭ 3.1 Ϯ 0.6 s-1 (n ϭ 3); for S573E, rOC(5 mM MgATP ϩ PKA) ϭ 2.2 Ϯ 0.3 s-1 (n ϭ 7). Login to comment
166 For S573E channels, rCO(5 mM MgATP ϩ PKA) ϭ 0.9 Ϯ 0.2 s-1 (n ϭ 7, total estimate) and 1 Ϯ 0.2 s-1 (n ϭ 4, best estimate). Login to comment
183 Patches contained one WT (A), K464A (B), or S573E (D) channel, or more than one D572N (C) channel. Login to comment
184 (E) Summary of mean (ϮSEM) ␶b values at 5 mM MgATP and 300 nM PKA (n ϭ 30, 21, 9, and 7 for WT, K464A, D572N, and S573E, respectively). Login to comment

[hide] The Walker B motif of the second nucleotide-bindin... Biochem J. 2007 Jan 15;401(2):581-6. Stratford FL, Ramjeesingh M, Cheung JC, Huan LJ, Bear CE
The Walker B motif of the second nucleotide-binding domain (NBD2) of CFTR plays a key role in ATPase activity by the NBD1-NBD2 heterodimer.
Biochem J. 2007 Jan 15;401(2):581-6., 2007-01-15 [PMID:16989640]

Abstract [show]
CFTR (cystic fibrosis transmembrane conductance regulator), a member of the ABC (ATP-binding cassette) superfamily of membrane proteins, possesses two NBDs (nucleotide-binding domains) in addition to two MSDs (membrane spanning domains) and the regulatory 'R' domain. The two NBDs of CFTR have been modelled as a heterodimer, stabilized by ATP binding at two sites in the NBD interface. It has been suggested that ATP hydrolysis occurs at only one of these sites as the putative catalytic base is only conserved in NBD2 of CFTR (Glu1371), but not in NBD1 where the corresponding residue is a serine, Ser573. Previously, we showed that fragments of CFTR corresponding to NBD1 and NBD2 can be purified and co-reconstituted to form a heterodimer capable of ATPase activity. In the present study, we show that the two NBD fragments form a complex in vivo, supporting the utility of this model system to evaluate the role of Glu1371 in ATP binding and hydrolysis. The present studies revealed that a mutant NBD2 (E1371Q) retains wild-type nucleotide binding affinity of NBD2. On the other hand, this substitution abolished the ATPase activity formed by the co-purified complex. Interestingly, introduction of a glutamate residue in place of the non-conserved Ser573 in NBD1 did not confer additional ATPase activity by the heterodimer, implicating a vital role for multiple residues in formation of the catalytic site. These findings provide the first biochemical evidence suggesting that the Walker B residue: Glu1371, plays a primary role in the ATPase activity conferred by the NBD1-NBD2 heterodimer.

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40 For S573E the following primers were used: reverse primer 5 -CTAGGTATCCAAAAGGCTCGTCTAATAAATACAA- AT-3 and forward primer 5 -ATTTGTATTTATTAGACGAGC- CTTTTGGATACCTAG-3 . Login to comment
77 The dose-response of TNP-ATP binding to the mutant NBDs: S573E and E1371Q was analysed using a single site binding algorithm and curve fitting by GraphPad Prism to determine Kd values. Login to comment
84 Similarly, the Walker B mutant proteins (S573E) and (E1371Q) bearing polyhistidine tags and an HA tag, in the case of E1371Q, were expressed in Sf9 cells using the baculovirus system (Figure 1B). Login to comment
86 Both the purified NBD1 mutant (S573E) and purified NBD2 mutant (E1371Q) bind the ATP analogue, TNP-ATP, with low micromolar affinities (Figure 2), similar to the affinities previously reported for wild-type NBD1 (11.6 µM) and NBD2 (10.6 µM) [14]. Login to comment

[hide] Requirements for efficient correction of DeltaF508... Cell. 2012 Jan 20;148(1-2):164-74. doi: 10.1016/j.cell.2011.11.023. Mendoza JL, Schmidt A, Li Q, Nuvaga E, Barrett T, Bridges RJ, Feranchak AP, Brautigam CA, Thomas PJ
Requirements for efficient correction of DeltaF508 CFTR revealed by analyses of evolved sequences.
Cell. 2012 Jan 20;148(1-2):164-74. doi: 10.1016/j.cell.2011.11.023., [PMID:22265409]

Abstract [show]
Misfolding of DeltaF508 cystic fibrosis (CF) transmembrane conductance regulator (CFTR) underlies pathology in most CF patients. F508 resides in the first nucleotide-binding domain (NBD1) of CFTR near a predicted interface with the fourth intracellular loop (ICL4). Efforts to identify small molecules that restore function by correcting the folding defect have revealed an apparent efficacy ceiling. To understand the mechanistic basis of this obstacle, positions statistically coupled to 508, in evolved sequences, were identified and assessed for their impact on both NBD1 and CFTR folding. The results indicate that both NBD1 folding and interaction with ICL4 are altered by the DeltaF508 mutation and that correction of either individual process is only partially effective. By contrast, combination of mutations that counteract both defects restores DeltaF508 maturation and function to wild-type levels. These results provide a mechanistic rationale for the limited efficacy of extant corrector compounds and suggest approaches for identifying compounds that correct both defective steps.

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No. Sentence Comment
70 Two 508-coupled mutants, D529F and S573E, dramatically increased the relative folding yield of NBD1 by 3.07- &#b1; 0.13-fold and 1.85- &#b1; 0.06-fold, respectively, a level comparable to that of the second-site suppressors identified in the STE6 chimera screen (Figure 3A). Login to comment
71 Thermal denaturation of purified D529F and S573E NBD1 revealed that S573E raised the Tm by 2 C-5 C relative to wild-type depending on ATP concentration (Figure S4B), indicating the improved folding yield in the complementation assay could be accounted for by increased stability of the native state (Figure 3B). Login to comment
108 The same two mutations that improved NBD1 folding yield, D529F and S573E, increased the maturation efficiency of CFTR (Figure 4A, yellow and magenta bars, respectively). Login to comment
115 Top 20 508-Coupled Positions within NBD1 Using Four Independent Statistical Methods ELSC McBASC OMES SCA 435 453 460 S466T S466T 468 470 472 473 473 474 474 L475Y L475Y L475Y F490L F490L W496V W496V W496V W496V 503 505 507 509 512 513 Y517I Y517I Y517I Y517I 520 520 521 C524A C524A C524A C524A L526A L526A L526A L526A D529F D529F D529F D529F D537F D537F 543 Y563V Y563V Y563V Y563V A566P A566P 569 569 S573E P574A P574A P574A F575T F575T 578 582 E583G E583G 587 591 595 598 602 604 604 604 H609T 617 630 630 640 The alignment of 493 sequences was used to calculate pairwise coupling scores using each method (ELSC, SCA, McBASC, and OMES). Login to comment
120 Interestingly, the two suppressors identified by the coupling analysis, D529F and S573E, had much more modest effects on NBD1 yield in the presence of the DF508 mutant as might be expected. Login to comment
127 The surface view A B I539T G550E R553M R555K 3M WT F S466T L475Y F490L W496V Y517I C524A L526A D529F D537F Y563V A566P S573E P574A F575T E583G H609T 0 1 2 3 Relative Yield NBD1 ( -gal.) 25 30 35 40 45 0.0 0.5 1.0 Temperature (C ) Relative Turbitity 0 1 2 3 4 -5 0 5 10 WT F I539T I539T F S573E R555K D529F Relative Yield NBD1 ( -gal.) Tm Figure 3. Login to comment
129 All mutations altered the relative yield relative to wild-type NBD1 as shown in the bar chart (&#b1; standard error of the mean [SEM], n = 9 except for WT, DF508, D529F, and S573E where n = 18). Login to comment
130 Two of the 508-coupled positions, D529F and S573E, increase the yield of NBD1. Login to comment
132 (B) Thermal denaturation of 5 mM purified recombinant WT, D529F, and S573E NBD1 in the presence of 2 mM ATP (left panel). Login to comment
134 S573E (magenta circles) increased the melting temperature 2 C. Login to comment
166 B C A B 0 1 2 3 0 1 2 Relative Yield NBD1 (b2;-gal.) Relative Yield CFTR (ELISA) WT ࢞F WT ƊF S466T L475Y F490L W496V Y517I C524A L526A D529F D537F Y563V A566P S573E P574A F575T E583G H609T 0 1 2 Relative Yield CFTR (ELISA) I539T G550E R553M R555K 3M Figure 4. Login to comment
168 (A) The efficiency of full-length CFTR maturation was determined by ELISA (&#b1;SEM, n = 6 for WT, DF508, D529F, and S573E, n = 3 for other mutants). Login to comment
171 The two 508-coupled positions that increased NBD1 folding yield, D529F and S573E, also increased the maturation yield of CFTR (yellow and magenta bars, respectively). Login to comment
173 The two F508- coupled position mutations, D529F and S573E, are colored in yellow and magenta circles, respectively. Login to comment
185 Previously identified second-site suppressor (I539T, G550E, R553M, R555K, and 3M) but not the 508-coupled mutants (D529F and S573E) increase the yield of DF508 NBD1. Login to comment
190 Identified in this study, D529F and S573E improve folding of NBD1 in isolation and maturation of full-length CFTR in the wild-type background. Login to comment
233 D529F, S573E, and R555K mutations of human wild-type NBD1 were expressed and purified as previously described (Thibodeau et al., 2005). Login to comment