ABCMdb

PMID: 16361259

Gross CH, Abdul-Manan N, Fulghum J, Lippke J, Liu X, Prabhakar P, Brennan D, Willis MS, Faerman C, Connelly P, Raybuck S, Moore J
Nucleotide-binding domains of cystic fibrosis transmembrane conductance regulator, an ABC transporter, catalyze adenylate kinase activity but not ATP hydrolysis.
J Biol Chem. 2006 Feb 17;281(7):4058-68. Epub 2005 Dec 16., 2006-02-17 [PubMed]

Sentences

No. Mutations Sentence Comment

10 ABCC7 p.Gly551Asp Interestingly, the G551D gating mutation has an exaggerated nonlinear phase compared with the wild type and may indicate this mutation affects the ability of NBD1 to dimerize. Login to comment 32 ABCC7 p.Gly551Asp In contrast, the second most common CF mutation G551D is only defective in channel function (6) and has a prevalence of 2-7% in the CF population (7). Login to comment 60 ABCC7 p.Gly551Asp The G551D gating mutation has an exaggerated nonlinear phase as compared with wild type, and may indicate this mutation affects NBD1 ability to form dimers. Login to comment 103 ABCC7 p.Gly551Asp The mNBD1 proteins (wild type and G551D) were purified to homogeneity as judged by a Coomassie-stained SDS-PAGE gel (Fig. 1A). Login to comment 104 ABCC7 p.Gly551Asp A total of nine mNBD1 wild type and four mNBD1 G551D independent protein samples were purified for this study. Login to comment 132 ABCC7 p.Gly551Asp A, Coomassie-stained SDS-PAGE of native mouse NBD1 (mNBD1) wild type and G551D mutant proteins (2 ␮g of protein). Login to comment 191 ABCC7 p.Lys1250Ala B, thin layer chromatography plateshowingtheAKactivityofthewild-typehNBD2 protein and the K1250A mutant protein. Login to comment 196 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp TABLE 1 Wild-type and mutant protein kinetic parameters for AK activity Enzymea Mutation ATP AMP ADP Km b,c Vmax b,c Km b,c Vmax b,c Km b,c Vmax b,c ␮M ␮M/min ␮M ␮M/min ␮M ␮M/min mNBD1 WT 30 Ϯ 2 1.2 Ϯ 0.01 30 Ϯ 4 0.9 Ϯ 0.02 450 Ϯ 60 1.4 Ϯ 0.06 mNBD1 G551D 80 Ϯ 7 0.4 Ϯ 0.02 70 Ϯ 7 0.4 Ϯ 0.02 360 Ϯ 50 1.0 Ϯ 0.05 hNBD1 WT 80 Ϯ 5 0.7 Ϯ 0.02 60 Ϯ 7 0.7 Ϯ 0.03 300 Ϯ 20 1.0 Ϯ 0.02 hNBD2 WT 70 Ϯ 3 0.6 Ϯ 0.01 40 Ϯ 5 0.7 Ϯ 0.03 280 Ϯ 20 0.7 Ϯ 0.02 a Enzyme concentrations are mNBD1 WT (900 nM), G551D (1800 nM), hNBD1 (4.7 nM), hNBD2 (30 nM). Login to comment 205 ABCC7 p.Lys1250Ala The hNBD2 (K1250A) mutant result is in agreement with the results reported by Randak and Welsh (16). Login to comment 240 ABCC7 p.Gly551Asp ABCC7 p.Lys464Ala Next, we wanted to examine three key CFTR residues in mNBD1 (K464A, G551D, and ⌬F508) to determine what effect these mutations had on their ability to form homodimers or alter AK activity. Login to comment 241 ABCC7 p.Lys464Ala All three mutant proteins overexpressed, however, during their purifications two mutants (⌬F508 and K464A) were problematic. Login to comment 244 ABCC7 p.Lys464Ala Because of the chaperone contamination we were forced to abandon our analysis of the K464A and ⌬F508 proteins. Login to comment 245 ABCC7 p.Gly551Asp In contrast, the mNBD1 G551D mutant protein purified with the same characteristics as the wild-type protein. Login to comment 246 ABCC7 p.Gly551Asp The AK activity of G551D mutant was measured as a function of protein concentration (Fig. 6A). Login to comment 247 ABCC7 p.Gly551Asp From this data, we find the G551D gating mutation lacks AK activity at low protein concentrations (Ͻ250 nM) when compared with the wild type protein, suggesting the mutation affects the ability of the NBD1 to dimerize. Login to comment 248 ABCC7 p.Gly551Asp This was confirmed with two independently purified mNBD1 G551D samples. Login to comment 249 ABCC7 p.Gly551Asp The kinetic parameters of mNBD1 G551D protein (1800 nM) were determined (Table 1) at saturating ATP (2 mM). Login to comment 250 ABCC7 p.Gly551Asp The apparent AMP Km was 70 ␮M and Vmax was 0.4 ␮M/min. At high AMP (400 ␮M) the apparent ATP Km was 80 ␮M and Vmax was 0.4 ␮M/min. At high protein concentrations, the G551D mutation results in only a modest decrease in catalysis and 2-fold decrease in substrate affinity when compared with wild type protein. Login to comment 251 ABCC7 p.Gly551Asp These data suggest the G551D mutation does not significantly disrupt catalytic function directly but rather the mutation may reduce the affinity between the two NBDs in the intact CFTR protein. Login to comment 253 ABCC7 p.Gly551Asp We surmise that the G551D mutation renders NBD1 unable to associate or rearrange productively with NBD2 upon nucleotide substrate binding and that this subsequently leads to a defect in Cl-ion transport. Login to comment 255 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp However, we cannot formally rule out the possibility that the CFTR G551D folds less efficiently than the wild-type protein (i.e. that there are a lower percentage of active molecules in G551D preparation as compared with wild type preparation) as a good active site titrating compound is not available. Login to comment 266 ABCC7 p.Gly551Asp A, mNBD1 wild type (square), mNBD1 G551D mutant (triangle) hNBD1 (B) and hNBD2 (C) proteins are shown. Login to comment