PMID: 16989640

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 [PubMed]
Sentences
No. Mutations Sentence Comment
6 ABCC7 p.Glu1371Gln
X
ABCC7 p.Glu1371Gln 16989640:6:49
status: NEW
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The present studies revealed that a mutant NBD2 (E1371Q) retains wild-type nucleotide binding affinity of NBD2. Login to comment
30 ABCC7 p.Lys1250Ala
X
ABCC7 p.Lys1250Ala 16989640:30:59
status: NEW
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ABCC7 p.Glu1371Ala
X
ABCC7 p.Glu1371Ala 16989640:30:94
status: NEW
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In support of this model, mutation of the Walker A lysine (K1250A) or the Walker B glutamate (E1371A/Q) in the conventional catalytic site leads to defective channel closure, resulting in prolonged channel open Abbreviations used: ABC, ATP-binding cassette; CF, cystic fibrosis; CFTR, CF transmembrane conductance regulator; HA, haemagglutinin; NBD, nucleotide-binding domain; MSD, membrane spanning domain; PFO, pentadecafluorooctanoic acid; TNP-ATP, 2 (3)-O-(2,4,6-trinitrophenyl)adenosine 5-triphosphate. Login to comment
34 ABCC7 p.Lys1250Ala
X
ABCC7 p.Lys1250Ala 16989640:34:67
status: NEW
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Although the consequences of mutating the Walker A lysine of NBD2 (K1250A) on nucleotide binding and hydrolysis have been measured [19], to date there have been no direct measurements of the consequences of mutating the putative catalytic base, Glu1371 , on nucleotide binding and hydrolysis. Login to comment
40 ABCC7 p.Ser573Glu
X
ABCC7 p.Ser573Glu 16989640:40:4
status: NEW
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For S573E the following primers were used: reverse primer 5 -CTAGGTATCCAAAAGGCTCGTCTAATAAATACAA- AT-3 and forward primer 5 -ATTTGTATTTATTAGACGAGC- CTTTTGGATACCTAG-3 . Login to comment
41 ABCC7 p.Glu1371Gln
X
ABCC7 p.Glu1371Gln 16989640:41:4
status: NEW
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For E1371Q, the reverse primer 5 - CAAATGAGCACTGGGTTGATCAAGCAGC-3 and the forward primer 5 -GCTGCTTGATCAACCCAGTGCTCATTTG-3 were used. Login to comment
65 ABCC7 p.Glu1371Gln
X
ABCC7 p.Glu1371Gln 16989640:65:84
status: NEW
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ATP depletion experiments Infected Sf9 cells expressing NBD1 and NBD2 (wild-type or E1371Q) were washed with PBS and collected by centrifugation. Login to comment
77 ABCC7 p.Ser573Glu
X
ABCC7 p.Ser573Glu 16989640:77:57
status: NEW
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ABCC7 p.Glu1371Gln
X
ABCC7 p.Glu1371Gln 16989640:77:67
status: NEW
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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 ABCC7 p.Ser573Glu
X
ABCC7 p.Ser573Glu 16989640:84:41
status: NEW
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ABCC7 p.Glu1371Gln
X
ABCC7 p.Glu1371Gln 16989640:84:53
status: NEW
view ABCC7 p.Glu1371Gln details
ABCC7 p.Glu1371Gln
X
ABCC7 p.Glu1371Gln 16989640:84:118
status: NEW
view ABCC7 p.Glu1371Gln details
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 ABCC7 p.Ser573Glu
X
ABCC7 p.Ser573Glu 16989640:86:31
status: NEW
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ABCC7 p.Glu1371Gln
X
ABCC7 p.Glu1371Gln 16989640:86:64
status: NEW
view ABCC7 p.Glu1371Gln details
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
88 ABCC7 p.Glu1371Gln
X
ABCC7 p.Glu1371Gln 16989640:88:25
status: NEW
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Walker B mutant of NBD2 (E1371Q) inhibits normal ATPase activity conferred by heterodimerization with NBD1 The ATPase activity of CFTR is conferred by heterodimerization of NBD1 and NBD2. Login to comment
164 ABCC7 p.Glu1371Gln
X
ABCC7 p.Glu1371Gln 16989640:164:153
status: NEW
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Interestingly, we could not detect a significant difference in the association between wild-type NBD1 and NBD2 and the association between NBD1 and NBD2(E1371Q) following ATP depletion in cell culture prior to cell lysis (with the removal of extracellular glucose, and the addition of deoxyglucose and rotenone). Login to comment
165 ABCC7 p.Glu1371Gln
X
ABCC7 p.Glu1371Gln 16989640:165:262
status: NEW
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In light of the recent studies by Vergani et al. [30] showing that ATP binding induced hydrogen bond formation between NBD1 and NBD2 in the vicinity of the 'catalytic site`, we predicted that the most stable interaction may have occurred between NBD1 and NBD2- (E1371Q) in ATP-replete cells. Login to comment