ABCMdb

PMID: 11919200

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., [PubMed]

Sentences

No. Mutations Sentence Comment

5 ABCA4 p.Gly863Ala ABCA4 p.Pro940Arg The G863A and P940R mutations were found to have significant attenuation of the rates of nucleotide hydrolysis and binding affinities. Login to comment 6 ABCA4 p.Arg943Gln On the other hand, the R943Q mutation had small, but detectable reduction in its nucleotidase activity and nucleotide binding affinity. Login to comment 136 ABCA4 p.Gly863Ala ABCA4 p.Arg943Gln ABCA4 p.Pro940Arg 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 ABCA4 p.Gly863Ala ABCA4 p.Arg943Gln ABCA4 p.Pro940Arg 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 139 ABCA4 p.Gly863Ala ABCA4 p.Arg943Gln ABCA4 p.Pro940Arg 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 ABCA4 p.Gly863Ala ABCA4 p.Arg943Gln ABCA4 p.Pro940Arg 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 142 ABCA4 p.Gly863Ala ATPase and CTPase activities of NBD1 wild-type and NBD1/G863A mutant proteins. Login to comment 143 ABCA4 p.Gly863Ala A, comparison of ATP hydrolysis by NBD1wt and NBD1/G863A polypeptides. Login to comment 144 ABCA4 p.Gly863Ala ABCA4 p.Gly863Ala Protein titration of purified NBD1 wild-type and NBD1/G863A proteins in a standard ATPase assay was carried out as described under "Materials and Methods" at 37 &#b0;C for 60 min using the indicate amounts of protein and ATP concentration of 500 òe;M. B, CTP hydrolysis by wtNBD1 and NBD1/G863A. Login to comment 145 ABCA4 p.Gly863Ala ABCA4 p.Gly863Ala ABCA4 p.Gly863Ala ATPase and CTPase activities of NBD1 wild-type and NBD1/G863A mutant proteins. Login to comment 146 ABCA4 p.Gly863Ala ABCA4 p.Gly863Ala A, comparison of ATP hydrolysis by NBD1wt and NBD1/G863A polypeptides. Login to comment 147 ABCA4 p.Gly863Ala ABCA4 p.Gly863Ala Protein titration of purified NBD1 wild-type and NBD1/G863A proteins in a standard ATPase assay was 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 NBD1/G863A. Login to comment 148 ABCA4 p.Gly863Ala ABCA4 p.Gly863Ala Protein titration of purified NBD1 wild-type and NBD1/G863A 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. 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/G863A proteins. Login to comment 149 ABCA4 p.Gly863Ala NBD1 wild-type (Ⅺ) and G863A mutant (E). Login to comment 150 ABCA4 p.Gly863Ala Consequences of the Gly-863 3 Ala Mutation on the ATPase/ CTPase Activity of NBD1 Protein-The ABCR mutation G863A is a commonly encountered mutation and has been reported in patients suffering from Stargardt disease (2, 9-11), age-related macular dystrophy (11), fundus flavimaculatus (5), and retinitis pigmentosa (10). Login to comment 152 ABCA4 p.Gly863Ala The results presented in Fig. 3A indicated that the ATPase function of G863A mutant protein was reduced b03;3-fold as compared with NBD1wt, indicating b03;70% of inhibition of the ATPase activity. Login to comment 153 ABCA4 p.Gly863Ala ABCA4 p.Gly863Ala Consequences of the Gly-863 3 Ala Mutation on the ATPase/ CTPase Activity of NBD1 Protein-The ABCR mutation G863A is a commonly encountered mutation and has been reported in patients suffering from Stargardt disease (2, 9-11), age-related macular dystrophy (11), fundus flavimaculatus (5), and retinitis pigmentosa (10). Login to comment 155 ABCA4 p.Gly863Ala The results presented in Fig. 3A indicated that the ATPase function of G863A mutant protein was reduced ϳ3-fold as compared with NBD1wt, indicating ϳ70% of inhibition of the ATPase activity. Login to comment 156 ABCA4 p.Gly863Ala ABCA4 p.Gly863Ala The Vmax (ATPase) for G863A mutant was 128 pmol/min/mg and that of the wild-type NBD1 was 584 pmol/min/mg (Table II). Login to comment 157 ABCA4 p.Gly863Ala In this case, the CTP hydrolysis of G863A was reduced to b03;30% of the activity of NDB1wt. Login to comment 158 ABCA4 p.Gly863Ala 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 159 ABCA4 p.Gly863Ala ABCA4 p.Pro940Arg However, in the mutant G863A protein the CTPase activity was reduced (Fig. 3B). Login to comment 160 ABCA4 p.Gly863Ala ABCA4 p.Pro940Arg In this case, the CTP hydrolysis of G863A was reduced to ϳ30% of the activity of NDB1wt. Login to comment 161 ABCA4 p.Gly863Ala ABCA4 p.Pro940Arg 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 ABCA4 p.Pro940Arg ABCA4 p.Pro940Arg 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 ABCA4 p.Pro940Arg ABCA4 p.Pro940Arg Analysis of the CTPase and ATPase activities of NBD1/ P940R mutant protein. Login to comment 164 ABCA4 p.Pro940Arg ABCA4 p.Pro940Arg ABCA4 p.Pro940Arg A, ATP hydrolysis by NBD1/P940R polypeptide in comparison to NBD1wt polypeptide. Login to comment 165 ABCA4 p.Pro940Arg ABCA4 p.Pro940Arg Protein titration of purified NBD1/P940R and NBD1 wild-type proteins in a standard ATPase assay. Login to comment 166 ABCA4 p.Pro940Arg ABCA4 p.Pro940Arg 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 ABCA4 p.Pro940Arg ABCA4 p.Pro940Arg 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 ABCA4 p.Arg943Gln ABCA4 p.Pro940Arg 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 ABCA4 p.Arg943Gln ABCA4 p.Pro940Arg NBD1 wild-type (Ⅺ) and P940R mutant (‚;). Login to comment 170 ABCA4 p.Arg943Gln The protein titration of purified NBD1/R943Q and NBD1 wild-type proteins was performed in a standard ATPase assay as described under "Materials and Methods." Login to comment 171 ABCA4 p.Arg943Gln ABCA4 p.Arg943Gln Nucleotidase activities of R943Q mutant protein. Login to comment 172 ABCA4 p.Arg943Gln ABCA4 p.Arg943Gln ABCA4 p.Arg943Gln A, comparison of ATP hydrolysis by NBD1wt and NBD1/R943Q polypeptides. Login to comment 173 ABCA4 p.Arg943Gln ABCA4 p.Arg943Gln The protein titration of purified NBD1/R943Q and NBD1 wild-type proteins was performed in a standard ATPase assay as described under "Materials and Methods." Login to comment 174 ABCA4 p.Arg943Gln The assays were carried out at 37 °C for 60 min using the indicate amounts of protein and ATP concentration of 500 ␮M. B, hydrolysis of CTP by wtNBD1 and the mutant-R943Q. Login to comment 175 ABCA4 p.Arg943Gln ABCA4 p.Arg943Gln ABCA4 p.Pro940Arg 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 176 ABCA4 p.Arg943Gln NBD1 wild-type (Ⅺ) and R943Q mutant (●). Login to comment 177 ABCA4 p.Pro940Arg 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 178 ABCA4 p.Pro940Arg The NBD1 P940R mutant protein had defects in both ATP and CTP hydrolysis (Fig. 4). Login to comment 179 ABCA4 p.Pro940Arg 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 180 ABCA4 p.Gly863Ala ABCA4 p.Pro940Arg ABCA4 p.Pro940Arg 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 181 ABCA4 p.Arg943Gln Arg-943 3 Gln Is the Least Influential on ATP and CTP Hydrolysis of NBD1wt Protein-The amino acid change R943Q has been described as a polymorphism, because it has been found in control populations (5, 9-11, 14). Login to comment 182 ABCA4 p.Pro940Arg 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 ABCA4 p.Gly863Ala ABCA4 p.Pro940Arg The P940R mutation severely affected the NBD1 function, although unlike G863A, the degree of inhibition was different for ATP and CTP. Login to comment 184 ABCA4 p.Arg943Gln Arg-943 3 Gln Is the Least Influential on ATP and CTP Hydrolysis of NBD1wt Protein-The amino acid change R943Q has been described as a polymorphism, because it has been found in control populations (5, 9-11, 14). Login to comment 185 ABCA4 p.Arg943Gln The Vmax for R943Q mutant was 392 pmol/min/mg, and that of the wild-type NBD1 was 584 pmol/min/mg (Table II). Login to comment 186 ABCA4 p.Arg943Gln The time-course analysis of ATPase activity (Fig. 5C), suggested that the ATPase activity of R943Q protein function was about 40% reduced with respect to that observed for NBD1wt. Login to comment 187 ABCA4 p.Arg943Gln The Vmax for R943Q mutant was 172 pmol/min/mg and that of the wild-type NBD1 was 376 pmol/min/mg (Table II). Login to comment 188 ABCA4 p.Arg943Gln ABCA4 p.Arg943Gln The Vmax for R943Q mutant was 392 pmol/min/mg, and that of the wild-type NBD1 was 584 pmol/min/mg (Table II). Login to comment 189 ABCA4 p.Arg943Gln The time-course analysis of ATPase activity (Fig. 5C), suggested that the ATPase activity of R943Q protein function was about 40% reduced with respect to that observed for NBD1wt. Login to comment 190 ABCA4 p.Arg943Gln The Vmax for R943Q mutant was 172 pmol/min/mg and that of the wild-type NBD1 was 376 pmol/min/mg (Table II). Login to comment 191 ABCA4 p.Arg943Gln Therefore, the CTPase activity of R943Q was reduced 2-fold compared with that observed with wild-type NBD1 (Fig. 5), indicating a higher inhibitory effect of this mutation on the CTPase (ϳ57%) than ATPase (ϳ12%) activity. Login to comment 193 ABCA4 p.Gly863Ala ABCA4 p.Pro940Arg In general, the G863A and P940R point mutations increased the Km values as follows: 80, 66 òe;M, respectively. Login to comment 194 ABCA4 p.Gly863Ala ABCA4 p.Gly863Ala ABCA4 p.Arg943Gln ABCA4 p.Arg943Gln ABCA4 p.Pro940Arg ABCA4 p.Pro940Arg 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 196 ABCA4 p.Gly863Ala ABCA4 p.Pro940Arg In general, the G863A and P940R point mutations increased the Km values as follows: 80, 66 ␮M, respectively. Login to comment 197 ABCA4 p.Gly863Ala ABCA4 p.Arg943Gln ABCA4 p.Pro940Arg 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 ABCA4 p.Gly863Ala ABCA4 p.Arg943Gln ABCA4 p.Pro940Arg 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 ABCA4 p.Gly863Ala ABCA4 p.Arg943Gln ABCA4 p.Pro940Arg 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 211 ABCA4 p.Gly863Ala ABCA4 p.Arg943Gln ABCA4 p.Pro940Arg 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 215 ABCA4 p.Gly863Ala ABCA4 p.Arg943Gln ABCA4 p.Pro940Arg NBD1 wild-type (ǧa;), mutant G863A (E), mutant P940R (Éa;), and mutant R943Q (cf;). Login to comment 218 ABCA4 p.Gly863Ala ABCA4 p.Arg943Gln ABCA4 p.Pro940Arg 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 219 ABCA4 p.Gly863Ala ABCA4 p.Gly863Ala ABCA4 p.Arg943Gln ABCA4 p.Pro940Arg ABCA4 p.Pro940Arg NBD1 wild-type (Ⅺ), mutant G863A (E), mutant P940R (‚), and mutant R943Q (ɜf;). Login to comment 220 ABCA4 p.Arg943Gln The binding capacity of R943Q was comparable to wild-type. Login to comment 222 ABCA4 p.Gly863Ala ABCA4 p.Arg943Gln ABCA4 p.Pro940Arg 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 ABCA4 p.Gly863Ala ABCA4 p.Pro940Arg Thus, the ATP binding affinity was impaired in the G863A mutant and to a lesser extent in the P940R mutant. Login to comment 224 ABCA4 p.Arg943Gln The binding capacity of R943Q was comparable to wild-type. Login to comment 227 ABCA4 p.Gly863Ala ABCA4 p.Arg943Gln ABCA4 p.Pro940Arg 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 228 ABCA4 p.Pro87Asn In our specific case, this region is comprised of Pro-87 to Asn-296 and aligned well with two similar ATP binding protein domains with available x-ray crystallographic atomic coordinates. Login to comment 231 ABCA4 p.Gly863Ala ABCA4 p.Arg943Gln ABCA4 p.Pro940Arg 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 232 ABCA4 p.Pro87Asn In our specific case, this region is comprised of Pro-87 to Asn-296 and aligned well with two similar ATP binding protein domains with available x-ray crystallographic atomic coordinates. Login to comment 239 ABCA4 p.Gly863Ala ABCA4 p.Arg943Gln ABCA4 p.Pro940Arg 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 242 ABCA4 p.Gly863Ala B, NBD1/G863A mutant (E). Login to comment 243 ABCA4 p.Gly863Ala ABCA4 p.Arg943Gln ABCA4 p.Pro940Arg ABCA4 p.Pro940Arg 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 244 ABCA4 p.Arg943Gln D, NBD1/R943Q mutant ({). Login to comment 246 ABCA4 p.Gly863Ala B, NBD1/G863A mutant (E). Login to comment 247 ABCA4 p.Pro940Arg C, NBD1/P940R mutant (‚). Login to comment 248 ABCA4 p.Arg943Gln D, NBD1/R943Q mutant ({). Login to comment 249 ABCA4 p.Gly863Ala ABCA4 p.Arg943Gln ABCA4 p.Pro940Arg 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 253 ABCA4 p.Gly863Ala ABCA4 p.Arg943Gln ABCA4 p.Pro940Arg 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 259 ABCA4 p.Gly863Ala The G863A mutation is a frequently occurring mutation and associated with a number of retinal diseases (Table I). Login to comment 261 ABCA4 p.Gly863Ala The G863A mutation in the NBD1 polypeptide resulted in a b03;70% decrease of the ATPase and CTPase activities of NBD1wt (Fig. 3). Login to comment 262 ABCA4 p.Gly863Ala The rate of ATP hydrolysis was considerably decreased; the observed Vmax of ATPase activity for the G863A mutant was 128 pmol/min/mg. Login to comment 263 ABCA4 p.Gly863Ala The G863A mutation is a frequently occurring mutation and associated with a number of retinal diseases (Table I). Login to comment 265 ABCA4 p.Gly863Ala The G863A mutation in the NBD1 polypeptide resulted in a ϳ70% decrease of the ATPase and CTPase activities of NBD1wt (Fig. 3). Login to comment 266 ABCA4 p.Gly863Ala ABCA4 p.Gly863Ala The rate of ATP hydrolysis was considerably decreased; the observed Vmax of ATPase activity for the G863A mutant was 128 pmol/min/mg. Login to comment 267 ABCA4 p.Gly863Ala Consequently, the G863A mutation appeared to lead to a general defect in nucleotide hydrolysis. Login to comment 268 ABCA4 p.Pro940Arg 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 ABCA4 p.Pro940Arg We have carried out a detailed kinetic analysis of P940R mutant of NBD1. Login to comment 270 ABCA4 p.Pro940Arg ABCA4 p.Gly963Ala The P940R mutant had a reduced nucleotidase activity comparable to that observed with G963A. Login to comment 271 ABCA4 p.Gly863Ala Protein titration analyses also showed that, although NBD1wt has ϳ3-fold higher CTPase than ATPase activity, the G863A appeared to affect the hydrolysis of both nucleotides to a similar extent. Login to comment 272 ABCA4 p.Gly863Ala ABCA4 p.Gly863Ala ABCA4 p.Pro940Arg Consequently, the G863A mutation appeared to lead to a general defect in nucleotide hydrolysis. Login to comment 273 ABCA4 p.Gly863Ala ABCA4 p.Gly863Ala ABCA4 p.Pro940Arg ABCA4 p.Pro940Arg 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 ABCA4 p.Gly863Ala ABCA4 p.Arg943Gln ABCA4 p.Pro940Arg ABCA4 p.Pro940Arg We have carried out a detailed kinetic analysis of P940R mutant of NBD1. Login to comment 275 ABCA4 p.Pro940Arg ABCA4 p.Gly963Ala The P940R mutant had a reduced nucleotidase activity comparable to that observed with G963A. Login to comment 276 ABCA4 p.Gly863Ala ABCA4 p.Arg943Gln ABCA4 p.Pro940Arg 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 277 ABCA4 p.Gly863Ala ABCA4 p.Gly863Ala ABCA4 p.Arg943Gln ABCA4 p.Pro940Arg ABCA4 p.Pro940Arg In general, both the G863A and P940R mutations were comparable in attenuating the nucleotidase activities. Login to comment 278 ABCA4 p.Gly863Ala ABCA4 p.Gly863Ala ABCA4 p.Pro940Arg 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 ABCA4 p.Gly863Ala ABCA4 p.Arg943Gln ABCA4 p.Pro940Arg 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 ABCA4 p.Gly863Ala ABCA4 p.Arg943Gln ABCA4 p.Pro940Arg 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 ABCA4 p.Gly863Ala ABCA4 p.Arg943Gln ABCA4 p.Arg943Gln ABCA4 p.Pro940Arg 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 283 ABCA4 p.Arg943Gln In fact, the dissociation constant for R943Q was lower than that of the wild-type. Login to comment 284 ABCA4 p.Gly863Ala ABCA4 p.Pro940Arg 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 ABCA4 p.Gly863Ala ABCA4 p.Arg943Gln ABCA4 p.Pro940Arg The overall order of nucleotide binding affinity was: R943Q b0e; wild-type b0e; P940R b0e; G863A. Login to comment 286 ABCA4 p.Gly863Ala ABCA4 p.Arg943Gln ABCA4 p.Pro940Arg 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 287 ABCA4 p.Arg943Gln The results indicated that dissociation constants for both wild-type and R943Q nonpathogenic mutation were comparable, and the values were 9.9 ϫ 10-7 M and 5 ϫ 10-7 M, respectively. Login to comment 288 ABCA4 p.Arg943Gln In fact, the dissociation constant for R943Q was lower than that of the wild-type. Login to comment 289 ABCA4 p.Gly863Ala ABCA4 p.Pro940Arg 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 ABCA4 p.Gly863Ala ABCA4 p.Arg943Gln ABCA4 p.Pro940Arg The overall order of nucleotide binding affinity was: R943Q Ͼ wild-type Ͼ P940R Ͼ G863A. Login to comment 291 ABCA4 p.Gly863Ala ABCA4 p.Arg943Gln ABCA4 p.Pro940Arg 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 295 ABCA4 p.Gly863Ala ABCA4 p.Pro940Arg 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 296 ABCA4 p.Gly863Ala The G863A mutation has been reported as one of the most frequently observed mutations in STGD patients in North America (2) and Netherlands (12). Login to comment 298 ABCA4 p.Gly863Ala ABCA4 p.Pro940Arg The P940R appeared to have a significant defect in nucleotide hydrolysis similar to that observed with G863A mutation. Login to comment 299 ABCA4 p.Pro940Arg The presence of the mutation P940R has been correlated with exudative age-related macular degeneration. Login to comment 300 ABCA4 p.Gly863Ala ABCA4 p.Pro940Arg 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 301 ABCA4 p.Gly863Ala ABCA4 p.Arg943Gln The G863A mutation has been reported as one of the most frequently observed mutations in STGD patients in North America (2) and Netherlands (12). Login to comment 303 ABCA4 p.Gly863Ala ABCA4 p.Pro940Arg The P940R appeared to have a significant defect in nucleotide hydrolysis similar to that observed with G863A mutation. Login to comment 304 ABCA4 p.Pro940Arg The presence of the mutation P940R has been correlated with exudative age-related macular degeneration. Login to comment 305 ABCA4 p.Gly863Ala ABCA4 p.Arg943Gln The R943Q mutation has also been shown to occur in conjunction with G863A leading to a more severe pathogenic state in humans (45). Login to comment 306 ABCA4 p.Arg943Gln The R943Q mutation displayed the minimal defects in nucleotide binding. Login to comment 308 ABCA4 p.Arg943Gln ABCA4 p.Pro940Arg 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 310 ABCA4 p.Gly863Ala ABCA4 p.Arg943Gln The R943Q mutation has also been shown to occur in conjunction with G863A leading to a more severe pathogenic state in humans (45). Login to comment 311 ABCA4 p.Pro940Arg 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 312 ABCA4 p.Arg943Gln In the case of the R943Q mutation, the change of a polar amino acid to a neutral amino acid may be less disruptive structurally. Login to comment 313 ABCA4 p.Arg943Gln ABCA4 p.Pro940Arg 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 ABCA4 p.Pro940Arg 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 317 ABCA4 p.Arg943Gln In the case of the R943Q mutation, the change of a polar amino acid to a neutral amino acid may be less disruptive structurally. Login to comment