ABCMdb

PMID: 15719171

Moran O, Galietta LJ, Zegarra-Moran O
Binding site of activators of the cystic fibrosis transmembrane conductance regulator in the nucleotide binding domains.
Cell Mol Life Sci. 2005 Feb;62(4):446-60., [PubMed]

Sentences

No. Mutations Sentence Comment

2 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp Using epithelia formed by cells stably transfected with wild-type or mutant (G551D, G1349D) CFTR, we estimated the apparent dissociation constant, KD, of a series of CFTR activators by measuring the increase in the apical membrane current. Login to comment 32 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp Materials and methods Cell cultures Fisher rat thyroid (FRT) cells expressing WT, G551D or G1349D CFTR were cultured on 60-mm petri dishes with Coon`s modified F12 containing 5% fetal bovine serum, 2 mM L-glutamine, 50 U/ml penicillin, 50 µg/ml streptomycin and 600 µg/ml zeocin, as previously described [18]. Login to comment 63 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp Compound WT G551D G1349D Apigenin(a) 3. Login to comment 64 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp 1 ± 0.6 (4) 19.6 ± 6.1 (3) - Genistein(a) 19.6 ± 2.5 (15) 114.2 ± 12 (3) 34.38 ± 2.21 (3) UCCF-023(b) 0.5 ± 0.2 (3) - - UCCF-029(b) 1.5 ± 0.5 (4) 31.3 ± 3.5 (3) 11.7 ± 3.1 (5) UCCF-030(b) 0.5 ± 0.1 (3) 4.7 ± 1.3 (2) - UCCF-853(b) 1.17 (1) - - C02(b) 3.2 ± 0.3 (2) - - C03(b) 1.2 (1) 7.2 ± 3 (2) - Act01(c) 0.4 ± 0.04 (5) - 0.6 ± 0.2 (3) Act03(c) 0.07 ± 0.02 (2) - - Act04(c) 0.4 ± 0.08 (4) not active up to 20 µM - Act05(c) 0.13 ± 0.01 (4) not active up to 5 µM - Act09(d) 1.95 ± 0.7 (3) not active up to 50 µM - Act11(d) 0.3 ± 0.08 (3) not active up to 20 µM - Act12(d) 1.9 ± 1.3 (3) - - Act13(d) 0.2 ± 0.03 (3) not active up to 20 µM - Act14(d) 0.2 ± 0.04 (2) - - Act17(d) 1.1 ± 0.5 (6) - - Data were obtained from measurements of Isc on FRT cell monolayers expressing WT or mutant (G551D or G1349D) CFTR. Login to comment 84 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp Results Electrophysiology We compared the effect of different CFTR activators on the WT and on mutations of the signature sequences of CFTR, the NBD1 mutant G551D, and the mirror NBD2 mutant G1349D. Login to comment 90 ABCC7 p.Gly551Asp Our data indicated, as already observed in previous experiments [18], that the genistein dose-response curve on G551D epithelia was shifted to the right with respect to WT protein (fig. 3A, B). Login to comment 91 ABCC7 p.Gly1349Asp We found that genistein also caused an Isc increase on the G1349D mutant (fig. 3A). Login to comment 92 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp The dose-response curve for the G1349D mutant was shifted to the right with respect to WT cells, but to a lesser extent than that of G551D (fig. 3B). Login to comment 94 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp In all cases, the affinities for the G551D mutant were significantly shifted to the right with respect to the WT protein, and the affinities for G1349D were in-between (fig. 3C, D). Login to comment 114 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp Effect of different CFTR activators on polarized epithelial preparations expressing WT or CFTR mutants (G1349D and G551D). Login to comment 117 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp Note that the lowest concentration that inhibited CFTR currents was 100 µM for WT and 200 µM for G1349D, while 200 µM still stimulated G551D. Login to comment 118 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp (B-D) Normalized dose-response relationships of FRT cells expressing WT (closed circles), G1349D (open triangles) and G551D (open squares) CFTR to genistein (B), UCCF-029 (C), and apigenin (D). Login to comment 170 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp The two mutations studied, G551D and G1349D, are located in the LSGGQ signature of NBD1 and NBD2, respectively. Login to comment 177 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp Hydrophobic (DGHB) and electrostatic (DGelec) contributions to interaction between NBD1 and NBD2 from WT and the mutants G551D and G1349D. Login to comment 178 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp WT G551D G1349D NBD1 + NBD2 ∆GHB (kJ/mol) -66.1 -68.4 -69.3 ∆Gelec (kJ/mol) -17.2 -12.5 -13.1 NBD1 + NBD2 + 2 ATP ∆GHB (kJ/mol) -98.4 -98.44 -101.05 ∆Gelec (kJ/mol) -28.9 -23.3 -24.3 Data were calculated from the interaction of NBDs in the absence of ATP, NBD1-NBD2, and from NBDs interacting in the presence of ATP, NBD1-NBD2-2 ATP. Login to comment 180 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp ABCC7 p.Gly1349Asp ABCC7 p.Gly1349Asp The hydrophobic contribution to the interaction energy is slightly improved in the mutants (DGHB = -68.4 kJ/mol for G551D, and DGHB = -69.3 kJ/mol for G1349D), but electrostatic terms of energy are significantly increased (DGElec = -12.5 kJ/mol for mutant G551D, and DGElec = -13.1 kJ/mol for mutant G1349D), with a net increase in the interaction energy of 2.9 kJ/mol and 1.4 kJ/mol for G551D and G1349D, respectively (table 2). Login to comment 183 ABCC7 p.Gly551Asp For mutant G551D, there is a reduction in the affinity for ATP in site 2 (DDGbind = 13 kJ/mol), but ATP-binding site 1 is almost unaffected (DDGbind = 1.83 kJ/mol). Login to comment 184 ABCC7 p.Gly1349Asp Conversely, for mutant G1349D, the affinity forATP is reduced in site 1 (DDGbind = 11.1 kJ/mol), with a smaller effect on site 2 (DDGbind = 3.3 kJ/mol). Login to comment 205 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp Comparison of the binding free energy differences, DGbind estimated for ATP-binding sites 1 and 2, for WT and the mutants G551D and G1349D of human CFTR. Login to comment 206 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp WT WT-G551D WT-G1349D DDGbind (kJ/mol) DDGbind (kJ/mol) Site 1 1.83 11.06 Site 2 12.98 3.3 Site1-site 2 -2.78 13.92 -4.99 Changes are expressed as differences in DGbind (DDGbind), calculated at different conditions. Login to comment 234 ABCC7 p.Gly551Asp Similarly, we estimated the KD for some of the compounds for which the affinity for the mutant G551D was too low to be measured experimentally (see table 1). Login to comment 242 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp Filled symbols represent data obtained docking the CFTR-activator to the WT model, open triangles are from G551D, and open circles are from G1349D. Login to comment 246 ABCC7 p.Gly551Asp Discussion We recently studied the effect of some CFTR activators on cells expressing either the WT or G551D mutant CFTR [18]. Login to comment 247 ABCC7 p.Gly551Asp We found that the genistein dose-response curve in G551D cells was shifted with respect to WT CFTR so that higher concentrations were required to observe activation. Login to comment 248 ABCC7 p.Gly551Asp This result strongly indicated that the G551D mutation is near a binding site for genistein. Login to comment 259 ABCC7 p.Gly551Asp Similar results have already been reported for genistein on the mutation G551D [18] and for genistein and two benzimidazolone analogs with the mutation dF508 [19]. Login to comment 260 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp Now, we extend these observation to five substances tested on mutant G551D and three tested on mutant G1349D. Login to comment 261 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp Interestingly, there is a decrease of affinity for every CFTR activator tested on the mutants, the reduction being more marked for mutant G551D than for mutant G1349D (table 1). Login to comment 292 ABCC7 p.Gly551Asp Indeed, comparing the empirical estimations of the binding free energy difference, we found that DDGbind for mutant G551D is consistent with a strong reduction inATP affinity at site 2, where the mutated residue is more directly involved, and a smaller reduction in ATP affinity for site 1. Login to comment 293 ABCC7 p.Gly1349Asp Similarly, as site 1 is more directly affected by mutation G1349D, a more severe reduction in ATP affinity is observed than in site 2. Login to comment 294 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp These results are in agreement with direct measurements of ATP binding done in NBDs of multidrug resistance protein-1 [31], where mutations G771D and G1433D of LSGGQ signatures, equivalent to G551D and G1349D in CFTR NBDs, produced similar effects on ATP-NBD interactions. Login to comment 371 ABCC7 p.Gly551Asp Acta 217: 23-28 18 Zegarra-Moran O., Romio L., Folli C., Caci E., Becq F., Vierfond J. M. et al. (2002) Correction of G551D-CFTR transport defect in epithelial monolayers by genistein but not by CPX or MPB-07. Login to comment