ABCC7 p.Lys464His
| ClinVar: |
c.1392G>T
,
p.Lys464Asn
?
, not provided
|
| Predicted by SNAP2: | A: D (95%), C: D (95%), D: D (95%), E: D (95%), F: D (95%), G: D (95%), H: D (95%), I: D (95%), L: D (95%), M: D (95%), N: D (91%), P: D (95%), Q: D (95%), R: D (95%), S: D (95%), T: D (95%), V: D (95%), W: D (95%), Y: D (95%), |
| Predicted by PROVEAN: | A: D, C: D, D: D, E: D, F: D, G: D, H: D, I: D, L: D, M: D, N: D, P: D, Q: D, R: D, S: D, T: D, V: D, W: D, Y: D, |
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[hide] Cystic fibrosis transmembrane conductance regulato... Biochem J. 2003 Apr 15;371(Pt 2):451-62. Annereau JP, Ko YH, Pedersen PL
Cystic fibrosis transmembrane conductance regulator: the NBF1+R (nucleotide-binding fold 1 and regulatory domain) segment acting alone catalyses a Co2+/Mn2+/Mg2+-ATPase activity markedly inhibited by both Cd2+ and the transition-state analogue orthovanadate.
Biochem J. 2003 Apr 15;371(Pt 2):451-62., 2003-04-15 [PMID:12523935]
Abstract [show]
Cystic fibrosis (CF) is caused by mutations in the gene encoding CFTR (cystic fibrosis transmembrane conductance regulator), a regulated anion channel and member of the ATP-binding-cassette transporter (ABC transporter) superfamily. Of CFTR's five domains, the first nucleotide-binding fold (NBF1) has been of greatest interest both because it is the major 'hotspot' for mutations that cause CF, and because it is connected to a unique regulatory domain (R). However, attempts have failed to obtain a catalytically active NBF1+R protein in the absence of a fusion partner. Here, we report that such a protein can be obtained following its overexpression in bacteria. The pure NBF1+R protein exhibits significant ATPase activity [catalytic-centre activity (turnover number) 6.7 min(-1)] and an apparent affinity for ATP ( K (m), 8.7 microM) higher than reported previously for CFTR or segments thereof. As predicted, the ATPase activity is inhibited by mutations in the Walker A motif. It is also inhibited by vanadate, a transition-state analogue. Surprisingly, however, the best divalent metal activator is Co(2+), followed by Mn(2+) and Mg(2+). In contrast, Ca(2+) is ineffective and Cd(2+) is a potent inhibitor. These novel studies, while demonstrating clearly that CFTR's NBF1+R segment can act independently as an active, vanadate-sensitive ATPase, also identify its unique cation activators and a new inhibitor, thus providing insight into the nature of its active site.
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No. Sentence Comment
44 To obtain ht-NBF1jR mutants in the Walker A region (K464H and K464A), cDNA cassettes were exchanged between BamHI and Bst1107I sites with homologous cDNA cassettes excised from the mutant pMALCR1 MBP-NBF1 plasmids, constructed previously in our laboratory [31].
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ABCC7 p.Lys464His 12523935:44:52
status: NEW48 Overexpression of the recombinant ht-NBF1TR (Gly-404-Lys-830) 'wild-type` and mutant proteins (K464H or K464A) Escherichia coli BL21 (DE3) strain, freshly transformed with the 'wild-type` (i.e. recombinant) and mutant ht-NBF1jR plasmids, were grown in 10 ml of LB medium containing 50 µg\ml kanamycin.
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ABCC7 p.Lys464His 12523935:48:95
status: NEW103 The Walker A motif is underlined, and Lys-464 within this motif is shown in bold to denote the site of mutations (K464H, K464A) made in this study.
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ABCC7 p.Lys464His 12523935:103:114
status: NEW105 (C) Expression in E. coli of the 'wild-type` ht-NBF1jR protein and mutant forms K464H and K464A.
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ABCC7 p.Lys464His 12523935:105:80
status: NEW109 Lanes 2, 4 and 6, E. coli cells transformed with plasmids containing, respectively, cDNA encoding 'wild-type` ht-NBF1jR, mutant-form K464H and mutant-form K464A prior to induction with IPTG; lanes 3, 5 and 7, as for lanes 2, 4 and 6 respectively but after induction with 0.3 mM IPTG.
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ABCC7 p.Lys464His 12523935:109:133
status: NEW110 (D) SDS/PAGE pattern obtained at different stages of purification of the 'wild-type` ht-NBF1jR protein and mutant forms K464H and K464A.
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ABCC7 p.Lys464His 12523935:110:120
status: NEW113 SDS/PAGE (10%) was used to analyse 10 µl aliquots at each step as follows: lane 1, molecular-mass markers as defined in (C); lanes 2-4, supernatant from guanidine hydrochloride-extracted E. coli overexpressing the 'wild-type` ht-NBF1jR protein and its mutant forms K464A and K464H respectively; lanes 5-7, flow-through fractions of the same extracts after their application to a Ni-NTA column (note that the $ 50 kDa proteins, i.e. the 'wild-type` ht-NBF1jR protein and its mutant forms, were retained); lanes 8-10, eluates derived from Ni-NTA columns containing, respectively, the 'wild-type` ht-NBF1jR protein and its K464A and K464H mutant forms after including 10 mM imidazole in the elution buffer; lanes 11-13, eluates derived from the same Ni-NTA columns after adding 200 mM imidazole to the elution buffer.
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ABCC7 p.Lys464His 12523935:113:280
status: NEWX
ABCC7 p.Lys464His 12523935:113:635
status: NEW120 E. coli transformed with the pET-28a(T) expression plasmid encoding NBF1TR residues 404-830, and a short His-tagged N-terminal region, markedly overproduce both 'wild-type` and mutant forms (K464H and K464A) of the protein Results presented in Figure 1(C) summarize SDS\PAGE patterns of E. coli cells harbouring the 'wild-type` and mutant ht-NBF1jR-containing plasmids before and after induction with IPTG.
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ABCC7 p.Lys464His 12523935:120:191
status: NEW122 The same applies to lane 5 (mutant K464H with IPTG) versus lane 4 (mutant K464H without IPTG), and to lane 7 (mutant K464A with IPTG) versus lane 6 (mutant K464A without IPTG).
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ABCC7 p.Lys464His 12523935:122:35
status: NEWX
ABCC7 p.Lys464His 12523935:122:74
status: NEW127 The ht-NBF1TR 'wild-type` protein and its mutant forms can be purified to apparent homogeneity on a Ni-NTA column and show cross-reactivity with a monoclonal antibody to CFTR exhibiting specificity for NBF1 As the overexpressed 'wild-type` and mutant (K464H and K464A) proteins were tagged with six histidines at their N-terminus (see the Materials and methods section and Figure 1B), they were amenable to purification on a column containing bound Ni#+.
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ABCC7 p.Lys464His 12523935:127:252
status: NEW128 For this reason, the wild-type and mutant E. coli cell pellets derived from the overexpression experiments described above were treated with a buffered solution containing the denaturant guanidine hydrochloride (6 M), and after centri- on the SDS/PAGE gels for the 'wild-type` ht-NBF1jR protein (lane 11) and its mutant forms K464A (lane 12) and K464H (lane 13).
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ABCC7 p.Lys464His 12523935:128:346
status: NEW152 (B) ATPase activity versus [ATP] plot for the purified renatured 'wild-type` ht-NBF1jR protein and mutant forms K464H and K464A.
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ABCC7 p.Lys464His 12523935:152:112
status: NEW154 The assay contained, in 75 µl of renaturation buffer, ATP concentrations of 0-100 µM, 5 mM MgCl2 and 0.47 µg of either the 'wild-type` ht-NBF1jR protein (WT) or its mutant forms K464H and K464A, pH 7.5.
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ABCC7 p.Lys464His 12523935:154:193
status: NEW159 (D) Relative ATPase activities obtained for the 'wild-type` NBF1jR Protein (WT) and mutant forms K464H and K464A using the [32 P]Pi release assay.
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ABCC7 p.Lys464His 12523935:159:97
status: NEW160 The assay was carried out in 200 µl of renaturation buffer, pH 7.5, containing 3.7 µg of the 'wild-type` ht-NBF1jR protein or its mutant forms (K464H, or K464A), and supplemented with 250 µM [γ-32 P]ATP (1.4i106 c.p.m.) and 1 mM divalent cation.
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ABCC7 p.Lys464His 12523935:160:154
status: NEW200 Subsequent studies carried out with ht-NBF1jR proteins with mutations in the Walker A consensus region (K464H and K464A) showed that the Vmax was reduced about 50% in each case (Figure 2B).
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ABCC7 p.Lys464His 12523935:200:104
status: NEW[hide] Cystic fibrosis: channel, catalytic, and folding p... J Bioenerg Biomembr. 1997 Oct;29(5):429-42. Seibert FS, Loo TW, Clarke DM, Riordan JR
Cystic fibrosis: channel, catalytic, and folding properties of the CFTR protein.
J Bioenerg Biomembr. 1997 Oct;29(5):429-42., [PMID:9511928]
Abstract [show]
The identification and characterization of the CFTR gene and protein have provided not only a major impetus to the dissection of the molecular pathophysiology of cystic fibrosis (CF) but also a new perspective on the structure and function of the large superfamily of membrane transport proteins to which it belongs. While the mechanism of the active vectorial translocation of many hydrophobic substrates by several of these transporters remains nearly as perplexing as it has for several decades, considerable insight has been gained into the control of the bidirectional permeation of chloride ions through a single CFTR channel by the phosphorylation of the R-domain and ATP interactions at the two nucleotide binding domains. However, details of these catalytic and allosteric mechanisms remain to be elucidated and await the replacement of two-dimensional conceptualizations with three dimensional structure information. Secondary and tertiary structure determination is required both for the understanding of the mechanism of action of the molecule and to enable a more complete appreciation of the misfolding and misprocessing of mutant CFTR molecules. This is the primary cause of the disease in the majority of the patients and hence understanding the details of the cotranslational interactions with multiple molecular chaperones, the ubiquitin-proteasome pathway and other components of the quality control machinery at the endoplasmic reticulum could provide a basis for the development of new therapeutic interventions.
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No. Sentence Comment
101 The observed Vmax of ~30 nmol/mg/min is very low compared to other ATPases such as the Ca++ -ATPase (600 nmol/mg/min; Racker, 1985) or P-glycoprotein (300-1650 nmol/mg/min; Sharom et al., 1995), although an indication of the significance of the data is given by a negative effect due to Walker A mutations K464H and K464L.
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ABCC7 p.Lys464His 9511928:101:306
status: NEW[hide] The first nucleotide binding fold of the cystic fi... J Biol Chem. 1995 Sep 22;270(38):22093-6. Ko YH, Pedersen PL
The first nucleotide binding fold of the cystic fibrosis transmembrane conductance regulator can function as an active ATPase.
J Biol Chem. 1995 Sep 22;270(38):22093-6., [PMID:7545672]
Abstract [show]
Cystic fibrosis is caused by mutations in the cell membrane protein called CFTR (cystic fibrosis transmembrane conductance regulator) which functions as a regulated Cl- channel. Although it is known that CFTR contains two nucleotide domains, both of which exhibit the capacity to bind ATP, it has not been demonstrated directly whether one or both domains can function as an active ATPase. To address this question, we have studied the first CFTR nucleotide binding fold (NBF1) in fusion with the maltose-binding protein (MBP), which both stabilizes NBF1 and enhances its solubility. Three different ATPase assays conducted on MBP-NBF1 clearly demonstrate its capacity to catalyze the hydrolysis of ATP. Significantly, the mutations K464H and K464L in the Walker A consensus motif of NBF1 markedly impair its catalytic capacity. MBP alone exhibits no ATPase activity and MBP-NBF1 fails to catalyze the release of phosphate from AMP or ADP. The Vmax of ATP hydrolysis (approximately 30 nmol/min/mg of protein) is significant and is markedly inhibited by azide and by the ATP analogs 2'-(3')-O-(2,4,6-trinitrophenyl)-adenosine-5'-triphosphate and adenosine 5'-(beta, gamma-imido)triphosphate. As inherited mutations within NBF1 account for most cases of cystic fibrosis, results reported here are fundamental to our understanding of the molecular basis of the disease.
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No. Sentence Comment
4 Significantly, the mutations K464H and K464L in the Walker A consensus motif of NBF1 markedly impair its catalytic capacity.
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ABCC7 p.Lys464His 7545672:4:29
status: NEW43 K464H: 5Ј-ACT GGA GCA GGC CAC ACT TCA CTT CTA-3Ј K464L: 5Ј-ACT GGA GCA GGC CTG ACT TCA CTT CTA-3Ј The identity of the two base changes was confirmed by DNA sequencing (20).
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ABCC7 p.Lys464His 7545672:43:0
status: NEW113 B, comparison of the purity of wild type and mutant MBP-NBF1 proteins (K464H and K464L).
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ABCC7 p.Lys464His 7545672:113:71
status: NEW115 C and D, the effect of the mutations K464H and K464L within NBF1 on the ATP hydrolytic activity of MBP-NBF1.
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ABCC7 p.Lys464His 7545672:115:37
status: NEW125 Even more convincing are results presented in Fig. 4, C and D, where it is seen that the mutations, K464H and K464L, in the Walker A nucleotide binding motif (GX4GKT) of NBF1 reduce the ATP hydrolytic capacity of purified mutant MBP-NBF1 proteins by over 80%.
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ABCC7 p.Lys464His 7545672:125:100
status: NEW133 The additional experimental results demonstrating that MBP alone has no catalytic capacity (Fig. 2C) and that mutations (K464H and K464L) within the Walker nucleotide binding motif GX4GKT markedly inhibit ATPase activity (Fig. 4, C and D) localize the catalytic site to NBF1.
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ABCC7 p.Lys464His 7545672:133:121
status: NEW42 K464H: 59-ACT GGA GCA GGC CAC ACT TCA CTT CTA-39 K464L: 59-ACT GGA GCA GGC CTG ACT TCA CTT CTA-39 The identity of the two base changes was confirmed by DNA sequencing (20).
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ABCC7 p.Lys464His 7545672:42:0
status: NEW112 B, comparison of the purity of wild type and mutant MBP-NBF1 proteins (K464H and K464L).
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ABCC7 p.Lys464His 7545672:112:71
status: NEW114 C and D, the effect of the mutations K464H and K464L within NBF1 on the ATP hydrolytic activity of MBP-NBF1.
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ABCC7 p.Lys464His 7545672:114:37
status: NEW124 Even more convincing are results presented in Fig. 4, C and D, where it is seen that the mutations, K464H and K464L, in the Walker A nucleotide binding motif (GX4GKT) of NBF1 reduce the ATP hydrolytic capacity of purified mutant MBP-NBF1 proteins by over 80%.
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ABCC7 p.Lys464His 7545672:124:100
status: NEW132 The additional experimental results demonstrating that MBP alone has no catalytic capacity (Fig. 2C) and that mutations (K464H and K464L) within the Walker nucleotide binding motif GX4GKT markedly inhibit ATPase activity (Fig. 4, C and D) localize the catalytic site to NBF1.
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ABCC7 p.Lys464His 7545672:132:121
status: NEW