ABCMdb

PMID: 16533065

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., [PubMed]

Sentences

No. Mutations Sentence Comment

36 ABCA4 p.Arg2038Trp ABCA4 p.Pro940Arg For example, the mutations P940R and R2038W are each localized to the nucleotide binding domains, NBD1 and NBD2, respectively. Login to comment 37 ABCA4 p.Pro940Arg The mutation P940R is associated with age-related macular degeneration, a late onset, slowly progressing disorder. Login to comment 38 ABCA4 p.Arg2038Trp On the other hand, R2038W has been observed in individuals afflicted with Stargardt macular dystrophy, an aggressive form of degeneration, striking individuals in early adulthood and rapidly leading to blindness. Login to comment 74 ABCA4 p.Arg2038Trp ABCA4 p.Lys969Ala ABCA4 p.Thr970Ala ABCA4 p.Thr1979Ala ABCA4 p.Lys1978Ala The following complementary oligonucleotides were used as mutagenic primers to generate the mutant NBD1 and NBD2 proteins: NBD1Pro940Arg (5'-GGT AAA GAT TTT TGA GCG CTG TGG CCG GCC AGC TG-3'), NBD1 Walker A, K969A, T970A (5'-CAA TGG AGC TGG GGC AGC CAC CAC CTT GTC CAT CC-3'), NBD2 Walker A, K1978A, T1979A (5'-GAA TGG TGC CGG CGC AGC AAC CAC ATT CAA GAT GC-3'), and NBD2 R2038W (5'-CTT TAC CTT TAT GCC AGG CTT CGA GGT GTA CCA GC-3'). Login to comment 111 ABCA4 p.Thr970Ala ABCA4 p.Thr1979Ala Lanes (from left to right): 1, NBD1WT; 2, NBD1P940R; 3, NBD1K969A,T970A; 4, NBD2WT; 5, NBD2R2038W; 6, NBD2K1978A,T1979A. Login to comment 154 ABCA4 p.Arg2038Trp The Mutation R2038W Is Associated with a Reduced Affinity of NBD1-NBD2 Interaction and the Loss of ADP Modulation. Login to comment 156 ABCA4 p.Arg2038Trp Earlier, we had carried out a detailed analysis of the dynamics of ATP binding and hydrolysis of the NBD2 polypeptide harboring the R2038W mutation. Login to comment 171 ABCA4 p.Arg2038Trp Attenuation of the ATPase ActiVity by Nucleotide Binding Domain Interaction Is Altered in the Stargardt Mutant R2038W. Login to comment 172 ABCA4 p.Arg2038Trp The NBD2 mutation, R2038W, leads to significant reductions in the rate of ATP hydrolysis as well as the affinity of ATP binding (67). Login to comment 173 ABCA4 p.Arg2038Trp To evaluate how this mutation affected the nucleotidase activity of the NBD1‚NBD2 complex, analysis of the ATPase activity of the R2038W mutant protein alone and in complex with wild-type NBD1 was carried out. Login to comment 177 ABCA4 p.Pro940Arg The Mutation P940R Leads to a Loss of NBD1 and NBD2 Interaction. Login to comment 178 ABCA4 p.Pro940Arg The mutation P940R has been reported in individuals with exudative age-related macular degeneration (AMD) (31, 32, 68). Login to comment 185 ABCA4 p.Pro940Arg The relative difference between Kd in the presence of ATP versus ADP was altered in the P940R mutant complex. Login to comment 187 ABCA4 p.Pro940Arg 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 ABCA4 p.Pro940Arg The Mutation P940R Does Not Alter Attenuation of ATPase ActiVity in the NBD1940‚NBD2 Complex. Login to comment 190 ABCA4 p.Pro940Arg The P940R mutation led to a decrease in ATPase activity of the NBD1 protein. Login to comment 193 ABCA4 p.Pro940Arg 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 198 ABCA4 p.Thr1979Ala A quantitative evaluation of the interaction between wild-type NBD1 and mutant NBD2K1978A,T1979A in the presence and absence of nucleotides was carried out (Table 1). Login to comment 199 ABCA4 p.Arg2038Trp ABCA4 p.Pro940Arg ABCA4 p.Lys969Ala ABCA4 p.Thr970Ala ABCA4 p.Lys1978Ala 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 202 ABCA4 p.Arg2038Trp The ATPase assay was carried out as described under Experimental Procedures at 37 °C for 60 min. A theoretical curve is shown, indicating the results expected if the activity of NBD1‚NBD2 R2038W were additive (]) of that obtained for the individual domains. Each point represents the average of three independent experiments. 1). Login to comment 218 ABCA4 p.Thr1979Ala ABCA4 p.Lys1978Ala On the other hand, the ratio of the experimental versus theoretical ATPase activities of the complex harboring NBD2 Walker A mutations, K1978A, T1979A, in the NBD2 domain was only 0.32, which represents a 68% difference in the experimental versus the theoretical activity, as compared to the 36% difference observed for the wild-type complex. Login to comment 224 ABCA4 p.Thr970Ala The ATPase assay was carried out as described under Experimental Procedures at 37 °C for 60 min. A theoretical curve is shown, indicating the results expected if the activity of NBD1P940R‚NBD2 were additive (]) of that obtained for the individual domains. Each point represents the average of three independent experiments. FIGURE 6: Effect of NBD1 Walker A mutations on modulation of ATPase of the NBD1‚NBD2 protein complex. Protein titration of the ATPase activity of purified NBD2WT (∆), NBD1K969A,T970A (0), NBD1WT (9), and the NBD1K969A,T970A‚NBD2 complex (O). Login to comment 225 ABCA4 p.Thr1979Ala ABCA4 p.Thr1979Ala ABCA4 p.Thr1979Ala ABCA4 p.Lys1978Ala The ATPase assay was carried out as described under Experimental Procedures at 37 °C for 60 min. A theoretical curve is shown, indicating the results expected if the activity of NBD1K969A,T970A‚ NBD2 were additive (]) of that obtained for the individual domains. Each point represents the average of three independent experiments. FIGURE 7: Effect of NBD2 Walker A mutations K1978A, T1979A on modulation of ATP hydrolysis of the NBD1‚NBD2 protein complex. Protein titration of the ATPase activity of purified NBD2K1978A,T1979A (∆), NBD2WT (2), NBD1WT (0) alone, and the NBD1‚NBD2K1978A,T1979A complex (O). Login to comment 226 ABCA4 p.Thr1979Ala The ATPase assay was carried out as described under Experimental Procedures at 37 °C for 60 min. A theoretical curve is shown, indicating the results expected if the activity of NBD1‚NBD2K1978A,T1979A were additive (]) of that obtained for the individual domains. Each point represents the average of three independent experiments. on NBD1. Login to comment 271 ABCA4 p.Arg2038Trp The only exception to this was with the NBD2 mutation R2038W, where mutation of this residue led to ATP hydrolysis of the complex that was nearly the sum of the individual NBDs, suggesting that Arg 2038 is critical to this inhibition. Login to comment