ABCMdb

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 The present studies revealed that a mutant NBD2 (E1371Q) retains wild-type nucleotide binding affinity of NBD2. Login to comment 30 ABCC7 p.Lys1250Ala ABCC7 p.Glu1371Ala 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 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 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 For E1371Q, the reverse primer 5 - CAAATGAGCACTGGGTTGATCAAGCAGC-3 and the forward primer 5 -GCTGCTTGATCAACCCAGTGCTCATTTG-3 were used. Login to comment 65 ABCC7 p.Glu1371Gln 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 ABCC7 p.Glu1371Gln 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 ABCC7 p.Glu1371Gln ABCC7 p.Glu1371Gln 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 ABCC7 p.Glu1371Gln 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 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 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 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