ABCA4 p.Pro940Arg
| ClinVar: |
c.2820C>G
,
p.Pro940=
?
, not provided
|
| Predicted by SNAP2: | A: N (53%), C: D (53%), D: D (53%), E: N (57%), F: D (66%), G: D (59%), H: N (57%), I: D (59%), K: N (57%), L: D (63%), M: D (59%), N: N (57%), Q: N (61%), R: N (53%), S: N (57%), T: N (61%), V: N (53%), W: D (80%), Y: D (59%), |
| Predicted by PROVEAN: | A: N, C: N, D: N, E: N, F: N, G: N, H: N, I: N, K: N, L: N, M: N, N: N, Q: N, R: N, S: N, T: N, V: N, W: N, Y: N, |
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Comments [show]
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[hide] Interaction of the nucleotide binding domains and ... Biochemistry. 2006 Mar 21;45(11):3813-23. Biswas-Fiss EE
Interaction of the nucleotide binding domains and regulation of the ATPase activity of the human retina specific ABC transporter, ABCR.
Biochemistry. 2006 Mar 21;45(11):3813-23., [PMID:16533065]
Abstract [show]
We report here a novel regulation of the ATPase activity of the human retina specific ATP binding cassette transporter (ABC), ABCR, by nucleotide binding domain interactions. We also present evidence that recombinant nucleotide binding domains of ABCR interact in vitro in the complete absence of transmembrane domains (TMDs). Although similar domain-domain interactions have been described in other ABC transporters, the roles of such interactions on the enzymatic mechanisms of these transporters have not been demonstrated experimentally. A quantitative analysis of the in vitro interactions as a function of the nucleotide-bound state demonstrated that the interaction takes place in the absence of nucleotide as well as in the presence of ATP and that it only attenuates in the ADP-bound state. Analysis of the ATPase activities of these proteins in free and complex states indicated that the NBD1-NBD2 interaction significantly influences the ATPase activity. Further investigation, using site-specific mutants, showed that mutations in NBD2 but not NBD1 led to the alteration of the ATPase activity of the NBD1.NBD2 complex and residue Arg 2038 is critical to this regulation. These data indicate that changes in the oligomeric state of the nucleotide binding domains of ABCR are coupled to ATP hydrolysis and might represent a possible signal for the TMDs of ABCR to export the bound substrate. Furthermore, the data support a mechanistic model in which, upon binding of NBD2, NBD1 binds ATP but does not hydrolyze it or does so with a significantly reduced rate.
Comments [show]
None has been submitted yet.
No. Sentence Comment
36 For example, the mutations P940R and R2038W are each localized to the nucleotide binding domains, NBD1 and NBD2, respectively.
X
ABCA4 p.Pro940Arg 16533065:36:27
status: NEW37 The mutation P940R is associated with age-related macular degeneration, a late onset, slowly progressing disorder.
X
ABCA4 p.Pro940Arg 16533065:37:13
status: NEW177 The Mutation P940R Leads to a Loss of NBD1 and NBD2 Interaction.
X
ABCA4 p.Pro940Arg 16533065:177:13
status: NEW178 The mutation P940R has been reported in individuals with exudative age-related macular degeneration (AMD) (31, 32, 68).
X
ABCA4 p.Pro940Arg 16533065:178:13
status: NEW185 The relative difference between Kd in the presence of ATP versus ADP was altered in the P940R mutant complex.
X
ABCA4 p.Pro940Arg 16533065:185:88
status: NEW187 However, in the case of P940R, the percent change between Kd in the presence of ATP versus ADP decreased to 38%.
X
ABCA4 p.Pro940Arg 16533065:187:24
status: NEW189 The Mutation P940R Does Not Alter Attenuation of ATPase ActiVity in the NBD1940‚NBD2 Complex.
X
ABCA4 p.Pro940Arg 16533065:189:13
status: NEW190 The P940R mutation led to a decrease in ATPase activity of the NBD1 protein.
X
ABCA4 p.Pro940Arg 16533065:190:4
status: NEW193 Consequently, the data indicate that the P940R mutation does not alter the nature of the modulation of ATPase activity observed in the NBD1P940R‚NBD2 complex, even though its own activity is substantially reduced.
X
ABCA4 p.Pro940Arg 16533065:193:41
status: NEW199 The affinity of interaction decreased under all conditions examined, as determined by comparison of the Kd values (Table Table 2: Modulation of ATPase Activities of NBD1‚NBD2 Complexes by Site-Specific Mutations NBD1‚NBD2 mutation domain NBD1‚NBD2 ATPasea (pmol/min) NBD1‚NBD2 ATPaseexptl (pmol/min) NBD1‚NBD2 ATPasecalcd b (pmol/min) motif/disease NBD1mut‚NBD2wt K969A, T970A NBD1 1.9 ( 0.4/5.3 ( 0.1 5.1 ( 0.1 7.2 synthetic NBD1wt‚NBD2mut K1978A, 1979A NBD2 3.1 ( 0.2/1.6 ( 0.3 1.5 ( 0.2 4.7 synthetic NBD1mut‚NBD2wt P940R NBD1 1.9 ( 0.3/5.2 ( 0.2 4.3 ( 0.1 7.1 AMD NBD1wt‚NBD2mut R2038W NBD2 3.1 ( 0.1/2.0 ( 0.2 4.4 ( 0.2 5.1 STGD NBD1wt‚NBD2wt wild type N/A 3.0 ( 0.3/5.4 ( 0.1 5.30 ( 0.3 8.4 none a Experimental ATPase activity corresponding to 6 pmol of the individual domains, NBD1 and NBD2, respectively.
X
ABCA4 p.Pro940Arg 16533065:199:575
status: NEW[hide] Biochemical defects in retina-specific human ATP b... J Biol Chem. 2002 Jun 14;277(24):21759-67. Epub 2002 Mar 27. Suarez T, Biswas SB, Biswas EE
Biochemical defects in retina-specific human ATP binding cassette transporter nucleotide binding domain 1 mutants associated with macular degeneration.
J Biol Chem. 2002 Jun 14;277(24):21759-67. Epub 2002 Mar 27., [PMID:11919200]
Abstract [show]
The retina-specific human ABC transporter (ABCR) functions in the retinal transport system and has been implicated in several inherited visual diseases, including Stargardt disease, fundus flavimaculatus, cone-rod dystrophy, and age-related macular degeneration. We have previously described a general ribonucleotidase activity of the first nucleotide binding domain (NBD1) of human ABCR (Biswas, E. E. (2001) Biochemistry 40, 8181-8187). In this communication, we present a quantitative study analyzing the effects of certain disease-associated mutations, Gly-863 --> Ala, Pro-940 --> Arg, and Arg-943 --> Gln on the nucleotide binding, and general ribonucleotidase activities of this domain. NBD1 proteins, harboring these mutations, were created through in vitro site-specific mutagenesis and expressed in Escherichia coli. Results of the enzyme-kinetic studies indicated that these mutations altered the ATPase and CTPase activities of NBD1. The G863A and P940R mutations were found to have significant attenuation of the rates of nucleotide hydrolysis and binding affinities. On the other hand, the R943Q mutation had small, but detectable reduction in its nucleotidase activity and nucleotide binding affinity. We have measured the nucleotide binding affinities of NBD1 protein and its mutants quantitatively by fluorescence anisotropy changes during protein binding to ethenoadenosine ATP (epsilonATP), a fluorescent ATP analogue. We have correlated the dissociation constant (K(D)) and the rates of nucleotide hydrolysis (V(max)) of NBD1 and its mutants with the available genetic data for these mutations.
Comments [show]
None has been submitted yet.
No. Sentence Comment
5 The G863A and P940R mutations were found to have significant attenuation of the rates of nucleotide hydrolysis and binding affinities.
X
ABCA4 p.Pro940Arg 11919200:5:14
status: NEW139 Equal amounts of cells before and after induction were analyzed by 5-18% SDS-PAGE followed by Coomassie Blue R-250 staining: lanes 1 and 2, BL21(DE3)/pET29aNBD1 wild-type cells before and after induction, respectively; lane 3, BL21(DE3)/pET29aNBD1/ G863A cells before induction, and after induction in lane 4; lanes 5 and 6, BL21(DE3)/pET29aNBD1/P940R cells before induction and after induction, respectively; lane 7, BL21(DE3)/pET29aNBD1/R943Q cells before induction and lane 8 after induction.
X
ABCA4 p.Pro940Arg 11919200:139:346
status: NEW141 Lane 1, wild-type NBD1 protein; lane 2, NBD1/G863A mutant protein; lane 3, NBD1/P940R mutant protein; and lane 4, NBD1/R943Q mutant protein.
X
ABCA4 p.Pro940Arg 11919200:141:80
status: NEW159 However, in the mutant G863A protein the CTPase activity was reduced (Fig. 3B).
X
ABCA4 p.Pro940Arg 11919200:159:101
status: NEW160 In this case, the CTP hydrolysis of G863A was reduced to ϳ30% of the activity of NDB1wt.
X
ABCA4 p.Pro940Arg 11919200:160:54
status: NEW161 The Vmax for G863A mutant was 104 pmol/min/mg and that of the wild-type NBD1 was 376 pmol/min/mg (Table II).
X
ABCA4 p.Pro940Arg 11919200:161:26
status: NEW162 Pro-940 3 Arg Mutation Abolishes the CTPase Function of Wild-type NBD1 Protein-The missense mutation P940R has been reported in patients with exudative age-related macular degeneration (14), which is the least frequent but most severe FIG. 4.
X
ABCA4 p.Pro940Arg 11919200:162:35
status: NEWX
ABCA4 p.Pro940Arg 11919200:162:101
status: NEW163 Analysis of the CTPase and ATPase activities of NBD1/ P940R mutant protein.
X
ABCA4 p.Pro940Arg 11919200:163:54
status: NEWX
ABCA4 p.Pro940Arg 11919200:163:215
status: NEW164 A, ATP hydrolysis by NBD1/P940R polypeptide in comparison to NBD1wt polypeptide.
X
ABCA4 p.Pro940Arg 11919200:164:26
status: NEWX
ABCA4 p.Pro940Arg 11919200:164:55
status: NEWX
ABCA4 p.Pro940Arg 11919200:164:302
status: NEW165 Protein titration of purified NBD1/P940R and NBD1 wild-type proteins in a standard ATPase assay.
X
ABCA4 p.Pro940Arg 11919200:165:35
status: NEWX
ABCA4 p.Pro940Arg 11919200:165:194
status: NEW166 The assays were carried out as described under "Materials and Methods" at 37 °C for 60 min using the indicate amounts of protein and ATP concentration of 500 M. B, CTP hydrolysis by wtNBD1 and the mutant P940R.
X
ABCA4 p.Pro940Arg 11919200:166:29
status: NEWX
ABCA4 p.Pro940Arg 11919200:166:217
status: NEW167 Protein titration of purified NBD1 wild-type and NBD1/ P940R proteins in a standard CTPase assay was carried out as described under "Materials and Methods" at 37 °C for 60 min using the indicate amounts of protein and CTP concentration of 500 M. C, time-course analysis of ATP hydrolysis by P940R mutant and wild-type protein.
X
ABCA4 p.Pro940Arg 11919200:167:55
status: NEWX
ABCA4 p.Pro940Arg 11919200:167:304
status: NEW168 Standard ATP assays were carried out as described under "Materials and Methods" at 37 °C for the times indicated using [␣-32 P]ATP and 2.5 g of purified NBD1 wild-type and NBD1/P940R proteins.
X
ABCA4 p.Pro940Arg 11919200:168:197
status: NEW169 NBD1 wild-type (Ⅺ) and P940R mutant (‚).
X
ABCA4 p.Pro940Arg 11919200:169:30
status: NEW175 Protein titration of purified NBD1 wild-type and NBD1/R943Q proteins in a standard CTPase assay was carried out as described under "Materials and Methods" at 37 °C for 60 min using the indicated amounts of protein and CTP concentration of 500 M. C, time-course analysis of ATP hydrolysis. Standard ATP assays were carried out as described under "Materials and Methods" at 37 °C for the times indicated using [␣-32 P]ATP and 2.5 g of purified wild-type and R943Q proteins.
X
ABCA4 p.Pro940Arg 11919200:175:9
status: NEW178 The NBD1 P940R mutant protein had defects in both ATP and CTP hydrolysis (Fig. 4).
X
ABCA4 p.Pro940Arg 11919200:178:9
status: NEW180 The CTPase activity in the P940R mutant protein was reduced to 84 pmol/min/mg whereas the ATPase activity was diminished to 129 pmol/min/mg (Table II).
X
ABCA4 p.Pro940Arg 11919200:180:4
status: NEWX
ABCA4 p.Pro940Arg 11919200:180:27
status: NEW182 The time-course analysis of ATP hydrolysis presented in Fig. 4C demonstrates that the rates of hydrolysis of both NBD1wt and P940R proteins were linear over 60 min before reaching equilibrium.
X
ABCA4 p.Pro940Arg 11919200:182:125
status: NEW183 The P940R mutation severely affected the NBD1 function, although unlike G863A, the degree of inhibition was different for ATP and CTP.
X
ABCA4 p.Pro940Arg 11919200:183:4
status: NEW196 In general, the G863A and P940R point mutations increased the Km values as follows: 80, 66 M, respectively.
X
ABCA4 p.Pro940Arg 11919200:196:26
status: NEW197 The rate of ATP hydrolysis was also altered and the Vmax values were 392 (R943Q), 129 (G863A), and 128 (P940R) pmol/min/mg.
X
ABCA4 p.Pro940Arg 11919200:197:104
status: NEW198 This represented a 78% decrease in Vmax for the G863A and P940R mutants, whereas the Vmax for R943Q mutant was diminished by only 33%.
X
ABCA4 p.Pro940Arg 11919200:198:58
status: NEW211 The plots, shown in Fig. 7, were generated by TABLE II Thermodynamic and kinetic parameters of ATP binding and 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 ATP binding ⌬G ° (kcal/mol) -8.2 -8.6 -8.1 -7.6 KD (M) 9.9 ϫ 10-7 5 ϫ 10-7 1.2 ϫ10-6 2.7 ϫ 10-6 Inhibition (%) ATP 0 33 78 78 CTP 0 54 78 72 Disease severity - ϩ/- ϩϩ ϩϩ FIG. 6.
X
ABCA4 p.Pro940Arg 11919200:211:187
status: NEW219 NBD1 wild-type (Ⅺ), mutant G863A (E), mutant P940R (‚), and mutant R943Q (ɜf;).
X
ABCA4 p.Pro940Arg 11919200:219:52
status: NEWX
ABCA4 p.Pro940Arg 11919200:219:94
status: NEW222 This nonlinear regression analysis gave dissociation constants (KD) for each of these proteins as follows: 9.9 ϫ 10-7 , 2.7 ϫ 10-6 , 1.2 ϫ 10-6 , and 5 ϫ 10-7 M, and ⌬G ϭ -8.2, -7.6, -8.1, and -8.6 kcal/mol for wild-type protein, G863A, P940R, and R943Q, respectively.
X
ABCA4 p.Pro940Arg 11919200:222:274
status: NEW223 Thus, the ATP binding affinity was impaired in the G863A mutant and to a lesser extent in the P940R mutant.
X
ABCA4 p.Pro940Arg 11919200:223:94
status: NEW231 We have generated a model for the NBD1 domain of ABCR (Fig. 9) using the Swiss-PDB and SYBYL6.7 homology-based protein structure modeling software to delineate the G863A, P940R, and R943Q mutations and analyze the possible structural implications on the wild-type NBD1 structure.
X
ABCA4 p.Pro940Arg 11919200:231:171
status: NEW243 Using refolded and highly purified and homogeneous preparations of wild-type, G863A, P940R, and R943Q NBD1 proteins (Fig. 2), we were able to examine the biochemical consequences of these mutations on the nucleotide binding and hydrolysis activity of FIG. 7.
X
ABCA4 p.Pro940Arg 11919200:243:8
status: NEWX
ABCA4 p.Pro940Arg 11919200:243:85
status: NEW247 C, NBD1/P940R mutant (‚).
X
ABCA4 p.Pro940Arg 11919200:247:8
status: NEW253 The data were fitted with BIOEQS using a simple binding model (monomer) for ⑀ATP binding to wild-type, G863A, P940R, and R943Q NBD1 proteins.
X
ABCA4 p.Pro940Arg 11919200:253:117
status: NEW272 Consequently, the G863A mutation appeared to lead to a general defect in nucleotide hydrolysis.
X
ABCA4 p.Pro940Arg 11919200:272:31
status: NEW273 The P940R mutation in ABCR has been linked with exudative age-related macular degeneration (14), which is a severe form of AMD.
X
ABCA4 p.Pro940Arg 11919200:273:4
status: NEWX
ABCA4 p.Pro940Arg 11919200:273:93
status: NEW274 We have carried out a detailed kinetic analysis of P940R mutant of NBD1.
X
ABCA4 p.Pro940Arg 11919200:274:51
status: NEWX
ABCA4 p.Pro940Arg 11919200:274:146
status: NEW275 The P940R mutant had a reduced nucleotidase activity comparable to that observed with G963A.
X
ABCA4 p.Pro940Arg 11919200:275:4
status: NEW277 In general, both the G863A and P940R mutations were comparable in attenuating the nucleotidase activities.
X
ABCA4 p.Pro940Arg 11919200:277:18
status: NEWX
ABCA4 p.Pro940Arg 11919200:277:31
status: NEW278 Perhaps the prevalence of the G863A mutation in the human population is much higher than the P940R, because exudative AMD is not a frequently encountered form of this disease, and as a result, the G863A mutation has been found to be associated with more common retinal diseases (44).
X
ABCA4 p.Pro940Arg 11919200:278:93
status: NEW279 The ATPase activity of the R943Q mutant protein was diminished, although the extent of diminution was not as great as that observed with G863A or P940R (Figs. 3-5, Table II).
X
ABCA4 p.Pro940Arg 11919200:279:146
status: NEW281 Overall, the enzymatic activity of mutants G863A and P940R was reduced more than that of the R943Q mutant, and the results on the nucleotide binding and hydrolysis correlate well with frequency and disease severity.
X
ABCA4 p.Pro940Arg 11919200:281:53
status: NEW282 Effects of G863A, P940R, and R943Q Mutations on the ⑀ATP Binding to NBD1 Protein-The biochemical effects in ATPase could be due to defects in nucleotide binding or hydrolysis or both.
X
ABCA4 p.Pro940Arg 11919200:282:18
status: NEW289 On the other hand, the dissociation constants for P940R and G863A pathogenic mutations were 1.2 ϫ 10-6 M and 2.7 ϫ 10-6 M, respectively.
X
ABCA4 p.Pro940Arg 11919200:289:50
status: NEW290 The overall order of nucleotide binding affinity was: R943Q Ͼ wild-type Ͼ P940R Ͼ G863A.
X
ABCA4 p.Pro940Arg 11919200:290:86
status: NEW291 The values for the free energy change involved in nucleotide binding (⌬G°) were as follows: -8.2 (wild-type), -8.6 (R943Q), -8.1 (P940R), and -7.6 (G863A) kcal/mole, respectively.
X
ABCA4 p.Pro940Arg 11919200:291:142
status: NEW300 Biochemical Defects in Mutants Appear to Be Related to the Disease Severity-According to our enzyme kinetic and fluorescence anisotropy results, the mutations that most severely affected both the ATPase activity and ⑀ATP binding of NBD1 were G863A and P940R.
X
ABCA4 p.Pro940Arg 11919200:300:259
status: NEW303 The P940R appeared to have a significant defect in nucleotide hydrolysis similar to that observed with G863A mutation.
X
ABCA4 p.Pro940Arg 11919200:303:4
status: NEW304 The presence of the mutation P940R has been correlated with exudative age-related macular degeneration.
X
ABCA4 p.Pro940Arg 11919200:304:29
status: NEW313 The model illustrates the spatial distribution of the amino acid changes of Pro-940 for arginine, and Arg-943 for glutamine, as well as the location of the Walker A and B motifs.
X
ABCA4 p.Pro940Arg 11919200:313:76
status: NEW316 In the mutation P940R, the change of a nonpolar amino acid (Pro) to a polar amino acid (Arg) would lead to a change in the orientation toward the surface of the protein because of the tendency of polar amino acids to be exposed to solvent.
X
ABCA4 p.Pro940Arg 11919200:316:16
status: NEW136 Equal amounts of cells before and after induction were analyzed by 5-18% SDS-PAGE followed by Coomassie Blue R-250 staining: lanes 1 and 2, BL21(DE3)/pET29aNBD1 wild-type cells before and after induction, respectively; lane 3, BL21(DE3)/pET29aNBD1/ G863A cells before induction, and after induction in lane 4; lanes 5 and 6, BL21(DE3)/pET29aNBD1/P940R cells before induction and after induction, respectively; lane 7, BL21(DE3)/pET29aNBD1/R943Q cells before induction and lane 8 after induction.
X
ABCA4 p.Pro940Arg 11919200:136:346
status: NEW138 Lane 1, wild-type NBD1 protein; lane 2, NBD1/G863A mutant protein; lane 3, NBD1/P940R mutant protein; and lane 4, NBD1/R943Q mutant protein.
X
ABCA4 p.Pro940Arg 11919200:138:80
status: NEW177 The CTPase activity in the P940R mutant protein was reduced to 84 pmol/min/mg whereas the ATPase activity was diminished to 129 pmol/min/mg (Table II).
X
ABCA4 p.Pro940Arg 11919200:177:27
status: NEW179 The time-course analysis of ATP hydrolysis presented in Fig. 4C demonstrates that the rates of hydrolysis of both NBD1wt and P940R proteins were linear over 60 min before reaching equilibrium.
X
ABCA4 p.Pro940Arg 11919200:179:125
status: NEW193 In general, the G863A and P940R point mutations increased the Km values as follows: 80, 66 òe;M, respectively.
X
ABCA4 p.Pro940Arg 11919200:193:26
status: NEW194 The rate of ATP hydrolysis was also altered and the Vmax values were 392 (R943Q), 129 (G863A), and 128 (P940R) pmol/min/mg. This represented a 78% decrease in Vmax for the G863A and P940R mutants, whereas the Vmax for R943Q mutant was diminished by only 33%.
X
ABCA4 p.Pro940Arg 11919200:194:104
status: NEWX
ABCA4 p.Pro940Arg 11919200:194:182
status: NEW207 The plots, shown in Fig. 7, were generated by TABLE II Thermodynamic and kinetic parameters of ATP binding and 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 ATP binding èc;G &#b0; (kcal/mol) afa;8.2 afa;8.6 afa;8.1 afa;7.6 KD (M) 9.9 afb; 10afa;7 5 afb; 10afa;7 1.2 afb;10afa;6 2.7 afb; 10afa;6 Inhibition (%) ATP 0 33 78 78 CTP 0 54 78 72 Disease severity afa; af9;/afa; af9;af9; af9;af9; FIG. 6.
X
ABCA4 p.Pro940Arg 11919200:207:187
status: NEW215 NBD1 wild-type (ǧa;), mutant G863A (E), mutant P940R (Éa;), and mutant R943Q (cf;).
X
ABCA4 p.Pro940Arg 11919200:215:51
status: NEW218 This nonlinear regression analysis gave dissociation constants (KD) for each of these proteins as follows: 9.9 afb; 10afa;7 , 2.7 afb; 10afa;6 , 1.2 afb; 10afa;6 , and 5 afb; 10afa;7 M, and èc;G afd; afa;8.2, afa;7.6, afa;8.1, and afa;8.6 kcal/mol for wild-type protein, G863A, P940R, and R943Q, respectively.
X
ABCA4 p.Pro940Arg 11919200:218:321
status: NEW227 We have generated a model for the NBD1 domain of ABCR (Fig. 9) using the Swiss-PDB and SYBYL6.7 homology-based protein structure modeling software to delineate the G863A, P940R, and R943Q mutations and analyze the possible structural implications on the wild-type NBD1 structure.
X
ABCA4 p.Pro940Arg 11919200:227:171
status: NEW239 Using refolded and highly purified and homogeneous preparations of wild-type, G863A, P940R, and R943Q NBD1 proteins (Fig. 2), we were able to examine the biochemical consequences of these mutations on the nucleotide binding and hydrolysis activity of FIG. 7.
X
ABCA4 p.Pro940Arg 11919200:239:85
status: NEW249 The data were fitted with BIOEQS using a simple binding model (monomer) for ঈATP binding to wild-type, G863A, P940R, and R943Q NBD1 proteins.
X
ABCA4 p.Pro940Arg 11919200:249:116
status: NEW268 The P940R mutation in ABCR has been linked with exudative age-related macular degeneration (14), which is a severe form of AMD.
X
ABCA4 p.Pro940Arg 11919200:268:4
status: NEW269 We have carried out a detailed kinetic analysis of P940R mutant of NBD1.
X
ABCA4 p.Pro940Arg 11919200:269:51
status: NEW270 The P940R mutant had a reduced nucleotidase activity comparable to that observed with G963A.
X
ABCA4 p.Pro940Arg 11919200:270:4
status: NEW276 Overall, the enzymatic activity of mutants G863A and P940R was reduced more than that of the R943Q mutant, and the results on the nucleotide binding and hydrolysis correlate well with frequency and disease severity.
X
ABCA4 p.Pro940Arg 11919200:276:53
status: NEW284 On the other hand, the dissociation constants for P940R and G863A pathogenic mutations were 1.2 afb; 10afa;6 M and 2.7 afb; 10afa;6 M, respectively.
X
ABCA4 p.Pro940Arg 11919200:284:50
status: NEW285 The overall order of nucleotide binding affinity was: R943Q b0e; wild-type b0e; P940R b0e; G863A.
X
ABCA4 p.Pro940Arg 11919200:285:86
status: NEW286 The values for the free energy change involved in nucleotide binding (èc;G&#b0;) were as follows: afa;8.2 (wild-type), afa;8.6 (R943Q), afa;8.1 (P940R), and afa;7.6 (G863A) kcal/mole, respectively.
X
ABCA4 p.Pro940Arg 11919200:286:158
status: NEW295 Defects in ABCR NBD1 Mutants Associated with Macular Degeneration Biochemical Defects in Mutants Appear to Be Related to the Disease Severity-According to our enzyme kinetic and fluorescence anisotropy results, the mutations that most severely affected both the ATPase activity and ঈATP binding of NBD1 were G863A and P940R.
X
ABCA4 p.Pro940Arg 11919200:295:326
status: NEW298 The P940R appeared to have a significant defect in nucleotide hydrolysis similar to that observed with G863A mutation.
X
ABCA4 p.Pro940Arg 11919200:298:4
status: NEW299 The presence of the mutation P940R has been correlated with exudative age-related macular degeneration.
X
ABCA4 p.Pro940Arg 11919200:299:29
status: NEW308 The model illustrates the spatial distribution of the amino acid changes of Pro-940 for arginine, and Arg-943 for glutamine, as well as the location of the Walker A and B motifs.
X
ABCA4 p.Pro940Arg 11919200:308:76
status: NEW311 In the mutation P940R, the change of a nonpolar amino acid (Pro) to a polar amino acid (Arg) would lead to a change in the orientation toward the surface of the protein because of the tendency of polar amino acids to be exposed to solvent.
X
ABCA4 p.Pro940Arg 11919200:311:16
status: NEW[hide] Genotype-phenotype analysis of ABCR variants in ma... Invest Ophthalmol Vis Sci. 2002 Feb;43(2):466-73. Bernstein PS, Leppert M, Singh N, Dean M, Lewis RA, Lupski JR, Allikmets R, Seddon JM
Genotype-phenotype analysis of ABCR variants in macular degeneration probands and siblings.
Invest Ophthalmol Vis Sci. 2002 Feb;43(2):466-73., [PMID:11818392]
Abstract [show]
PURPOSE: Single-copy variants of the autosomal recessive Stargardt disease (STGD1) gene ABCR (ABCA4) have been shown to confer enhanced susceptibility to age-related macular degeneration (AMD). To investigate the role of ABCR alleles in AMD further, genotype-phenotype analysis was performed on siblings of patients with AMD who had known ABCR variants. This genetically related population provides a cohort of subjects with similar age and ethnic background for genotype-phenotype comparison to the original probands. METHODS: All available siblings of 26 probands carrying probable disease-associated ABCR variants were examined clinically. Blood samples were collected from these siblings for genotype analysis to search for the ABCR variant alleles corresponding to the isofamilial proband. RESULTS: Nineteen of 33 siblings from 15 families carried the respective proband's variant ABCR allele. Some families exhibited concordance of ABCR alleles with macular degeneration phenotype, but others did not. Exudative AMD was uncommon among both probands and siblings. CONCLUSIONS: Although population studies have indicated that some ABCR variant alleles may enhance susceptibility to AMD, investigation of the extent of ABCR involvement by kindred analysis is complicated by a plethora of environmental and other hereditary factors not investigated in the current study that may also play important roles.
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No. Sentence Comment
54 Subsequent to the publication of the initial study, one of the original 167 patients was found to have a P940R ABCR variant that was not present in the STGD or general-population cohorts.
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ABCA4 p.Pro940Arg 11818392:54:105
status: NEW[hide] ABCR gene analysis in familial exudative age-relat... Invest Ophthalmol Vis Sci. 2000 Jan;41(1):244-7. Souied EH, Ducroq D, Rozet JM, Gerber S, Perrault I, Munnich A, Coscas G, Soubrane G, Kaplan J
ABCR gene analysis in familial exudative age-related macular degeneration.
Invest Ophthalmol Vis Sci. 2000 Jan;41(1):244-7., [PMID:10634626]
Abstract [show]
PURPOSE: Identification of genetic factors in the pathogenesis of age-related macular degeneration (AMD) is of crucial importance in this common cause of blindness. Mutations in the Stargardt disease gene (ABCR) were previously reported in patients with atrophic forms of AMD. The purpose of this study was to analyze familial segregation of ABCR gene mutations in 52 unrelated multiplex cases of exudative AMD. METHODS: A complete ophthalmological examination including visual acuity measurement, fundus examination, and fluorescein angiography (FA) was performed on each exudative AMD patient. The entire coding sequence of the ABCR gene was analyzed using a combination of single-strand conformation polymorphism and confirmatory sequencing of the exons showing an abnormal pattern of migration. RESULTS: Six heterozygous missense changes were identified. A lack of familial segregation was observed in 4 of 6 codon changes (Arg943Gln, Val1433Ile, Pro1948Leu, and Ser2255Ile). Conversely, 2 codon changes cosegregated with the disease in 2 small families: Pro940Arg and Leu1970Phe. CONCLUSIONS: The authors believe that segregation of the ABCR gene mutations with familial cases of AMD has not yet been shown. The analysis of familial segregation allowed the authors to exclude 4 of 6 codon changes as disease-causing mutations. Furthermore, it was shown here that the ABCR gene may be rarely involved in exudative AMD, with at best 2 of 52 familial cases (4%) related to this susceptibility factor.
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9 Conversely, 2 codon changes cosegregated with the disease in 2 small families: Pro940Arg and Leu1970Phe.
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ABCA4 p.Pro940Arg 10634626:9:79
status: NEW40 The heterozygous codon changes observed were Pro940Arg, Arg943Gln (2 families), Pro1948Leu (2 families), Leu1970Phe, Val1433Ile, and Ser2255Ile.
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ABCA4 p.Pro940Arg 10634626:40:45
status: NEW42 Conversely, the Pro940Arg, Val1433Ile, and Leu1970Phe changes were not found in the control group.
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ABCA4 p.Pro940Arg 10634626:42:16
status: NEW48 An association of the mutations with the disease was observed in the 2 small families harboring 2 mutations absent in controls: Pro940Arg and Leu1970Phe (Fig.
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ABCA4 p.Pro940Arg 10634626:48:128
status: NEW56 Pedigrees of the 2 families presenting the Pro940Arg and Leu1970Phe codon changes.
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ABCA4 p.Pro940Arg 10634626:56:43
status: NEW69 Second, for Pro940Arg and Leu1970Phe we observed possible segregation with AMD. However, the families were too small to draw any definite conclusions about causality because only one generation could be analyzed.
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ABCA4 p.Pro940Arg 10634626:69:12
status: NEW72 The Pro940Arg and the Leu1970Phe codon changes occur in codons adjacent to the first and second ATP-binding sites, respectively.
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ABCA4 p.Pro940Arg 10634626:72:4
status: NEW74 The Leu1970Phe mutation has been observed in compound heterozygous Stargardt patients so that this mutation is presumed to induce important ABCR protein alterations.18 This mutation was also previously identified in one case of AMD but not observed in the general population (n ϭ 220).19 To the best of our knowledge, the Pro940Arg mutation has not been reported previously.
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ABCA4 p.Pro940Arg 10634626:74:328
status: NEW[hide] Retinoid binding properties of nucleotide binding ... J Biol Chem. 2012 Dec 28;287(53):44097-107. doi: 10.1074/jbc.M112.409623. Epub 2012 Nov 9. 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., [PMID:23144455]
Abstract [show]
The retina-specific ATP binding cassette transporter, ABCA4 protein, is associated with a broad range of inherited macular degenerations, including Stargardt disease, autosomal recessive cone rod dystrophy, and fundus flavimaculatus. In order to understand its role in retinal transport in rod out segment discs, we have investigated the interactions of the soluble domains of ABCA4 with both 11-cis- and all-trans-retinal. Using fluorescence anisotropy-based binding analysis and recombinant polypeptides derived from the amino acid sequences of the four soluble domains of ABCA4, we demonstrated that the nucleotide binding domain 1 (NBD1) specifically bound 11-cis-retinal. Its affinity for all-trans-retinal was markedly reduced. Stargardt disease-associated mutations in this domain resulted in attenuation of 11-cis-retinal binding. Significant differences in 11-cis-retinal binding affinities were observed between NBD1 and other cytoplasmic and lumenal domains of ABCA4. The results suggest a possible role of ABCA4 and, in particular, the NBD1 domain in 11-cis-retinal binding. These results also correlate well with a recent report on the in vivo role of ABCA4 in 11-cis-retinal transport.
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102 Lane 1, WT-NBD1; lane 2, mutant P940R; lane 3, mutant R943Q; lane 4, mutant G863A.
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ABCA4 p.Pro940Arg 23144455:102:32
status: NEW171 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.
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ABCA4 p.Pro940Arg 23144455:171:179
status: NEW174 Three ABCA4 mutations associated with Stargardt disease (R943Q, P940R, and G863A) were chosen for analysis in this study.
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ABCA4 p.Pro940Arg 23144455:174:64
status: NEW178 We have explored the effects of missense mutations R943Q, P940R, and G863A on 11-cis-retinal interaction with the NBD1.
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ABCA4 p.Pro940Arg 23144455:178:58
status: NEW181 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).
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ABCA4 p.Pro940Arg 23144455:181:190
status: NEW182 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.
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ABCA4 p.Pro940Arg 23144455:182:78
status: NEW198 C, titration of 100 nM 11-cis-retinal with mutant P940R.
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ABCA4 p.Pro940Arg 23144455:198:50
status: NEW215 Three missense mutations in NBD1 were examined in this study, R943Q, P940R, and G863A.
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ABCA4 p.Pro940Arg 23144455:215:69
status: NEW220 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.
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ABCA4 p.Pro940Arg 23144455:220:13
status: NEW