ABCMdb

ABCA4 p.Pro940Arg

None has been submitted yet.

Publications

PMID: 16533065 [PubMed] Biswas-Fiss EE et al: "Interaction of the nucleotide binding domains and regulation of the ATPase activity of the human retina specific ABC transporter, ABCR."

No. Sentence Comment

36 For example, the mutations P940R and R2038W are each localized to the nucleotide binding domains, NBD1 and NBD2, respectively. Login to comment
37 The mutation P940R is associated with age-related macular degeneration, a late onset, slowly progressing disorder. Login to comment
177 The Mutation P940R Leads to a Loss of NBD1 and NBD2 Interaction. Login to comment
178 The mutation P940R has been reported in individuals with exudative age-related macular degeneration (AMD) (31, 32, 68). Login to comment
185 The relative difference between Kd in the presence of ATP versus ADP was altered in the P940R mutant complex. Login to comment
187 However, in the case of P940R, the percent change between Kd in the presence of ATP versus ADP decreased to 38%. Login to comment
189 The Mutation P940R Does Not Alter Attenuation of ATPase ActiVity in the NBD1940‚NBD2 Complex. Login to comment
190 The P940R mutation led to a decrease in ATPase activity of the NBD1 protein. Login to comment
193 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. Login to comment
199 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. Login to comment

PMID: 11919200 [PubMed] Suarez T et al: "Biochemical defects in retina-specific human ATP binding cassette transporter nucleotide binding domain 1 mutants associated with macular degeneration."

No. Sentence Comment

5 The G863A and P940R mutations were found to have significant attenuation of the rates of nucleotide hydrolysis and binding affinities. Login to comment
139 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. Login to comment
141 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. Login to comment
159 However, in the mutant G863A protein the CTPase activity was reduced (Fig. 3B). Login to comment
160 In this case, the CTP hydrolysis of G863A was reduced to ϳ30% of the activity of NDB1wt. Login to comment
161 The Vmax for G863A mutant was 104 pmol/min/mg and that of the wild-type NBD1 was 376 pmol/min/mg (Table II). Login to comment
162 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. Login to comment
163 Analysis of the CTPase and ATPase activities of NBD1/ P940R mutant protein. Login to comment
164 A, ATP hydrolysis by NBD1/P940R polypeptide in comparison to NBD1wt polypeptide. Login to comment
165 Protein titration of purified NBD1/P940R and NBD1 wild-type proteins in a standard ATPase assay. Login to comment
166 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. Login to comment
167 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. Login to comment
168 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. Login to comment
169 NBD1 wild-type (Ⅺ) and P940R mutant (‚). Login to comment
175 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. Login to comment
178 The NBD1 P940R mutant protein had defects in both ATP and CTP hydrolysis (Fig. 4). Login to comment
180 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). Login to comment
182 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. Login to comment
183 The P940R mutation severely affected the NBD1 function, although unlike G863A, the degree of inhibition was different for ATP and CTP. Login to comment
196 In general, the G863A and P940R point mutations increased the Km values as follows: 80, 66 ␮M, respectively. Login to comment
197 The rate of ATP hydrolysis was also altered and the Vmax values were 392 (R943Q), 129 (G863A), and 128 (P940R) pmol/min/mg. Login to comment
198 This represented a 78% decrease in Vmax for the G863A and P940R mutants, whereas the Vmax for R943Q mutant was diminished by only 33%. Login to comment
211 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. Login to comment
219 NBD1 wild-type (Ⅺ), mutant G863A (E), mutant P940R (‚), and mutant R943Q (ɜf;). Login to comment
222 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. Login to comment
223 Thus, the ATP binding affinity was impaired in the G863A mutant and to a lesser extent in the P940R mutant. Login to comment
231 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. Login to comment
243 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. Login to comment
247 C, NBD1/P940R mutant (‚). Login to comment
253 The data were fitted with BIOEQS using a simple binding model (monomer) for ⑀ATP binding to wild-type, G863A, P940R, and R943Q NBD1 proteins. Login to comment
272 Consequently, the G863A mutation appeared to lead to a general defect in nucleotide hydrolysis. Login to comment
273 The P940R mutation in ABCR has been linked with exudative age-related macular degeneration (14), which is a severe form of AMD. Login to comment
274 We have carried out a detailed kinetic analysis of P940R mutant of NBD1. Login to comment
275 The P940R mutant had a reduced nucleotidase activity comparable to that observed with G963A. Login to comment
277 In general, both the G863A and P940R mutations were comparable in attenuating the nucleotidase activities. Login to comment
278 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). Login to comment
279 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). Login to comment
281 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. Login to comment
282 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. Login to comment
289 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. Login to comment
290 The overall order of nucleotide binding affinity was: R943Q Ͼ wild-type Ͼ P940R Ͼ G863A. Login to comment
291 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. Login to comment
300 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. Login to comment
303 The P940R appeared to have a significant defect in nucleotide hydrolysis similar to that observed with G863A mutation. Login to comment
304 The presence of the mutation P940R has been correlated with exudative age-related macular degeneration. Login to comment
313 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. Login to comment
316 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. Login to comment
136 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. Login to comment
138 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. Login to comment
177 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). Login to comment
179 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. Login to comment
193 In general, the G863A and P940R point mutations increased the Km values as follows: 80, 66 òe;M, respectively. Login to comment
194 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%. Login to comment
207 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. Login to comment
215 NBD1 wild-type (ǧa;), mutant G863A (E), mutant P940R (Éa;), and mutant R943Q (cf;). Login to comment
218 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. Login to comment
227 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. Login to comment
239 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. Login to comment
249 The data were fitted with BIOEQS using a simple binding model (monomer) for ঈATP binding to wild-type, G863A, P940R, and R943Q NBD1 proteins. Login to comment
268 The P940R mutation in ABCR has been linked with exudative age-related macular degeneration (14), which is a severe form of AMD. Login to comment
269 We have carried out a detailed kinetic analysis of P940R mutant of NBD1. Login to comment
270 The P940R mutant had a reduced nucleotidase activity comparable to that observed with G963A. Login to comment
276 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. Login to comment
284 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. Login to comment
285 The overall order of nucleotide binding affinity was: R943Q b0e; wild-type b0e; P940R b0e; G863A. Login to comment
286 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. Login to comment
295 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. Login to comment
298 The P940R appeared to have a significant defect in nucleotide hydrolysis similar to that observed with G863A mutation. Login to comment
299 The presence of the mutation P940R has been correlated with exudative age-related macular degeneration. Login to comment
308 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. Login to comment
311 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. Login to comment

PMID: 11818392 [PubMed] Bernstein PS et al: "Genotype-phenotype analysis of ABCR variants in macular degeneration probands and siblings."

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. Login to comment

PMID: 10634626 [PubMed] Souied EH et al: "ABCR gene analysis in familial exudative age-related macular degeneration."

No. Sentence Comment

9 Conversely, 2 codon changes cosegregated with the disease in 2 small families: Pro940Arg and Leu1970Phe. Login to comment
40 The heterozygous codon changes observed were Pro940Arg, Arg943Gln (2 families), Pro1948Leu (2 families), Leu1970Phe, Val1433Ile, and Ser2255Ile. Login to comment
42 Conversely, the Pro940Arg, Val1433Ile, and Leu1970Phe changes were not found in the control group. Login to comment
48 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. Login to comment
56 Pedigrees of the 2 families presenting the Pro940Arg and Leu1970Phe codon changes. Login to comment
69 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. Login to comment
72 The Pro940Arg and the Leu1970Phe codon changes occur in codons adjacent to the first and second ATP-binding sites, respectively. Login to comment
74 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. Login to comment

PMID: 23144455 [PubMed] Biswas-Fiss EE et al: "Retinoid binding properties of nucleotide binding domain 1 of the Stargardt disease-associated ATP binding cassette (ABC) transporter, ABCA4."

No. Sentence Comment

102 Lane 1, WT-NBD1; lane 2, mutant P940R; lane 3, mutant R943Q; lane 4, mutant G863A. Login to comment
171 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 Three ABCA4 mutations associated with Stargardt disease (R943Q, P940R, and G863A) were chosen for analysis in this study. Login to comment
178 We have explored the effects of missense mutations R943Q, P940R, and G863A on 11-cis-retinal interaction with the NBD1. Login to comment
181 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 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
198 C, titration of 100 nM 11-cis-retinal with mutant P940R. Login to comment
215 Three missense mutations in NBD1 were examined in this study, R943Q, P940R, and G863A. Login to comment
220 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