ABCMdb

PMID: 23144455

Biswas-Fiss EE, Affet S, Ha M, Biswas SB
Retinoid binding properties of nucleotide binding domain 1 of the Stargardt disease-associated ATP binding cassette (ABC) transporter, ABCA4.
J Biol Chem. 2012 Dec 28;287(53):44097-107. doi: 10.1074/jbc.M112.409623. Epub 2012 Nov 9., [PubMed]

Sentences

No. Mutations Sentence Comment

102 ABCA4 p.Gly863Ala ABCA4 p.Arg943Gln ABCA4 p.Pro940Arg Lane 1, WT-NBD1; lane 2, mutant P940R; lane 3, mutant R943Q; lane 4, mutant G863A. Login to comment 171 ABCA4 p.Gly863Ala ABCA4 p.Arg943Gln ABCA4 p.Pro940Arg Error bars, S.D. TABLE 1 Thermodynamic and kinetic parameters of 11-cis-retinal binding and nucleotide hydrolysis for wild-type and mutant NBD1 proteins Parameter Wild type R943Q P940R G863A Enzyme kinetics Vmax (pmol/min/mg) ATP 584 392 129 128 CTP 376 172 84 104 Retinal binding 11-cis-Retinal Kd (M) 8.0 afe; 1.3 afb; 10afa;8 8.0 afe; 2.0 afb; 10afa;6 4.0 afe; 1.4 afb; 10afa;6 c56;1.0 afb; 10afa;5 11-cis-Retinal af9; 1.0 mM AMP-PNP Kd (M) 4.1 afe; 0.6 afb; 10afa;6 NDa ND ND 11-cis-Retinal af9; 1.0 mM ADP Kd (M) 3.6 afe; 0.8 afb; 10afa;7 ND ND ND -Fold inhibition NAb 100 50 NA Disease severity af9;/afa; af9;af9; af9;af9; a ND, not detectable with a ᐵ11-cis-retinalᐶ of c55;1 afb; 10afa;5 M. b NA, not applicable. Login to comment 174 ABCA4 p.Gly863Ala ABCA4 p.Arg943Gln ABCA4 p.Pro940Arg Three ABCA4 mutations associated with Stargardt disease (R943Q, P940R, and G863A) were chosen for analysis in this study. Login to comment 178 ABCA4 p.Gly863Ala ABCA4 p.Arg943Gln ABCA4 p.Pro940Arg We have explored the effects of missense mutations R943Q, P940R, and G863A on 11-cis-retinal interaction with the NBD1. Login to comment 180 ABCA4 p.Gly863Ala The binding of mutant G863A was significantly attenuated as indicated by the drastic right shift of the binding isotherm as well as its inability to achieve saturation in the presence of a high concentration of protein (Fig. 6A). Login to comment 181 ABCA4 p.Arg943Gln ABCA4 p.Pro940Arg Nonlinear regression analysis gave Kd of 8.0 afe; 1.3 afb; 10afa;8 M, 8.0 afe; 2.0 afb; 10afa;6 M, and 4.0 afe; 1.4 afb; 10afa;6 M for the wild type and R943Q and P940R mutations, respectively (Fig. 6 and Table 1). Login to comment 182 ABCA4 p.Arg943Gln ABCA4 p.Pro940Arg As a consequence of Stargardt disease mutations, 100-fold (R943Q) to 50-fold (P940R) decreases in the binding affinity of the NBD1 domain for 11-cis-retinal were observed. Login to comment 183 ABCA4 p.Gly863Ala The retinal binding of the G863A mutant was severely attenuated, and the Kd was c56;1.0 afb; 10afa;5 M. DISCUSSION The ABCA4 protein is an essential component of the visual transduction cycle of vertebrate retina. Login to comment 196 ABCA4 p.Gly863Ala A, titration of 100 nM 11-cis-retinal with mutant G863A. Login to comment 197 ABCA4 p.Arg943Gln B, titration of 100 nM 11-cis-retinal with mutant R943Q. Login to comment 198 ABCA4 p.Pro940Arg C, titration of 100 nM 11-cis-retinal with mutant P940R. Login to comment 215 ABCA4 p.Gly863Ala ABCA4 p.Arg943Gln ABCA4 p.Pro940Arg Three missense mutations in NBD1 were examined in this study, R943Q, P940R, and G863A. Login to comment 219 ABCA4 p.Gly863Ala The most significant change was observed with G863A, in which saturation binding was no longer observed. Login to comment 220 ABCA4 p.Arg943Gln ABCA4 p.Pro940Arg The mutation P940R seemed to affect 11-cis-retinal interaction the least and led to a b03;50-fold decrease in binding affinity, whereas the R943Q mutation led to a 100-fold decrease FIGURE 7. Login to comment