ABCMdb

PMID: 12962493

Biswas-Fiss EE
Functional analysis of genetic mutations in nucleotide binding domain 2 of the human retina specific ABC transporter.
Biochemistry. 2003 Sep 16;42(36):10683-96., [PubMed]

Sentences

No. Mutations Sentence Comment

73 ABCA4 p.Asp2177Asn ABCA4 p.Leu1971Arg ABCA4 p.Arg2038Trp ABCA4 p.Gly2146Asp ABCA4 p.Lys2175Ala The NBD2 expression vector pET29aNBD2 was used as template, 12 cycles of PCR, and each cycle was 30 s at 95 °C, 30 s at 50 °C, and 15 min at 68°C using complimentary oligonucleotides to produce the mutations L1971R, R2038W, G2146D, K2175A, and D2177N. Login to comment 74 ABCA4 p.Asp2177Asn ABCA4 p.Leu1971Arg ABCA4 p.Arg2038Trp ABCA4 p.Gly2146Asp ABCA4 p.Lys2175Ala ABCA4 p.Lys2175Ala The primers used for mutagenesis were as follows: L1971R, CGC CCT GGA GAG TGC TTT GGC CTC CGG GGA GTG AAT GGT GCC GGC AAA AC; R2038W, CTT TAC CTT TAT GCC AGG CTT CGA GGT GTA CCA GC, G2146D, CTG GCC ATC ATG GTA AAG GAC GCC TTT CGA TGT AT; D2177N, ATC AAA TCC CCG AAG GAC AAC CTG CTT CCT GAC CTG AAC; K2175A, CA ATG AAG ATC AAA TCC CCG GCG GAC GAC CTG CTT CCT GA. All of the mutations were disease-associated with the exception of K2175A. Login to comment 76 ABCA4 p.Leu2027Phe The pET29aNBD2 construct harboring the L2027F mutation was already available in our laboratory and prepared as previously described (12, 13). Login to comment 103 ABCA4 p.Asp2177Asn ABCA4 p.Leu1971Arg ABCA4 p.Arg2038Trp ABCA4 p.Leu2027Phe ABCA4 p.Gly2146Asp The locations of the disease associated mutations investigated in this study; L1971R, R2038W, G2146D, L2027F, and D2177N are indicated. Login to comment 144 ABCA4 p.Asp2177Asn ABCA4 p.Leu1971Arg ABCA4 p.Arg2038Trp ABCA4 p.Leu2027Phe ABCA4 p.Gly2146Asp ABCA4 p.Lys2175Ala Here, we have used site-specific mutagenesis to create disease-related genetic mutations: L1971R, D2177N, L2027F, R2038W, and G2146D as well as a synthetic mutation, K2175A. Login to comment 153 ABCA4 p.Asp2177Asn ABCA4 p.Leu1971Arg ABCA4 p.Arg2038Trp ABCA4 p.Leu2027Phe ABCA4 p.Gly2146Asp ABCA4 p.Lys2175Ala Lane 1: protein molecular weight standards; lane 2, wild-type NBD2; lane 3, L2027F mutant; lane 4, L1971R mutant; lane 5, D2177N; lane 6, G2146D; lane 7, R2038W mutant; lane 8, K2175A. Login to comment 165 ABCA4 p.Lys2175Ala Consequently, we generated a synthetic mutation K2175A to explore this hypothesis Effects of Genetic Mutations in the NBD2 Domain on Its ATPase ActiVity. Login to comment 168 ABCA4 p.Leu1971Arg The amino acid change, L1971R, is associated with mild to moderate forms of macular degeneration (Fundus flavimaculatus) (24). Login to comment 169 ABCA4 p.Leu1971Arg Analysis of NBD2 polypeptides harboring this mutation demonstrated that its effect on ATP hydrolysis was significant (i.e., a 57% decrease in specific activity with respect to the wild type control), and the observed rates of hydrolysis for L1971R (72 nmol/min/mg) were attenuated in comparison to the wild-type NBD2 (Figure 5, Table 1). Login to comment 176 ABCA4 p.Gly2146Asp In the mutation G2146D, the specific ATPase activity was reduced to approximately 70% of that observed with the wild type, and the rate of hydrolysis (65 nm/min/mg) was 56% of the wild-type NBD2. Login to comment 177 ABCA4 p.Arg2038Trp The mutation R2038W also led to a similar level of decrease in ATP hydrolysis relative to the wild-type control. Login to comment 179 ABCA4 p.Arg2038Trp ABCA4 p.Gly2146Asp The G2146D and R2038W mutations are associated with cone-rod dystrophy and Stargardt disease, respectively. These two degenerative syndromes are more severe than FFM or AMD, with respect to age of onset and time frame of disease progression. Login to comment 180 ABCA4 p.Asp2177Asn Unlike most NBD2 mutants, mutant NBD2 D2177N protein appears to have a higher specific activity and rate relative to the wild type (Figure 5). Login to comment 181 ABCA4 p.Asp2177Asn The D2177N mutation leads to hyperactivity of the enzyme as opposed to hypo- and null ABCR mutant phenotypes more frequently observed with NBD2 mutations (18). Login to comment 186 ABCA4 p.Asp2177Asn The substitution of a polar group for an acidic group in the mutation D2177N led to a small increase in the rate of hydrolysis (130 nmol/ min/mg) (Table 1). Login to comment 188 ABCA4 p.Lys2175Ala We created a site-specific mutant, K2175A, to explore this hypothesis. Login to comment 189 ABCA4 p.Lys2175Ala K2175A is not a naturally occurring disease-associated mutation. Login to comment 190 ABCA4 p.Lys2175Ala Analyses of the putative salt-bridge mutant, K2175A, demonstrated that this change led to the loss of ATPase activity. Login to comment 191 ABCA4 p.Lys2175Ala The lack of detectable levels of ATPase activity precluded kinetic analysis of the K2175A mutant. Login to comment 192 ABCA4 p.Lys2175Ala The K2175A mutation, as inferred from the homology-based model, leads to a significant disruption of the overall charge balance of the local environment, resulting in a net-negative charge in the region because of the presence of D2177 and D2176. Login to comment 218 ABCA4 p.Asp2177Asn ABCA4 p.Lys2175Ala The ATP binding constants for D2177N and K2175A were determined similarly (Figures 6B,E). Login to comment 220 ABCA4 p.Asp2177Asn ABCA4 p.Asp2177Asn ABCA4 p.Lys2175Ala ABCA4 p.Lys2175Ala The Kd for D2177N was 2.3 × 10-6 M, while that of K2175A was 1.1 × 10-6 M. These Kd values represented 4- and 2-fold decrease, from that observed in the wild type, in the binding affinity for D2177N and K2175A, respectively. Login to comment 222 ABCA4 p.Lys2175Ala In both of these mutants, particularly in the case of K2175A, alteration in ATP binding was minor. Login to comment 224 ABCA4 p.Lys2175Ala In fact, the free energy change associated with K2175A‚ ATP was close to that observed for the wild-type NBD2‚ ATP and the binding affinity of K2175A indicates that the significant loss of ATPase activity was not due to alteration in ATP binding. Login to comment 225 ABCA4 p.Leu1971Arg The binding affinity of mutant L1971R decreased approximately 4-fold from that of the wild type and Kd was determined to be 2.1 × 10-6 M (Figure 6A). Login to comment 227 ABCA4 p.Arg2038Trp ABCA4 p.Gly2146Asp The binding constants for both G2146D and R2038W decreased as compared to the wild-type control (Figure 6C,D). Login to comment 228 ABCA4 p.Arg2038Trp ABCA4 p.Gly2146Asp The affinity for ATP was quite similar for both mutants; the G2146D Kd was 1.33 × 10-6 M while that of R2038W was 1.32 × 10-6 M (Table 1). Login to comment 231 ABCA4 p.Leu2027Phe Previously, we reported that the STGD1-associated mutant L2027F resulted in a significantly impaired in ATP hydrolysis (12). Login to comment 232 ABCA4 p.Leu2027Phe Here, we have examined any possible changes in the nucleotide binding by the L2027F mutant. Login to comment 233 ABCA4 p.Leu2027Phe Fluorescence anisotropy analysis demonstrated that L2027F was defective in ATP binding, as saturation of binding was not observed (Figure 6F, Table 1). Login to comment 253 ABCA4 p.Asp2177Asn ABCA4 p.Leu1971Arg ABCA4 p.Arg2038Trp ABCA4 p.Gly2146Asp ABCA4 p.Lys2175Ala The curves represent a least squares nonlinear regression curve fit of the data representing the (A) L1971R mutant, (B) D2177N mutant, (C) G2146D mutant, (D) R2038W mutant, and (E) K2175A mutant. Login to comment 259 ABCA4 p.Leu1971Arg We observed only slight differences in the quenching profile of the L1971R mutant, both in the presence and in the absence of nucleotide binding (Figure 7B, Table 1). Login to comment 260 ABCA4 p.Leu1971Arg The L1971R mutation is associated with a "milder form" of macular degeneration, Fundus Flavimaculatus. Login to comment 261 ABCA4 p.Leu1971Arg The L1971R mutant was observed to have a reduced rate of ATP hydrolysis, 62% that of the wild type. Login to comment 263 ABCA4 p.Leu2027Phe Significant changes were observed in the Stern-Volmer plot for the L2027F mutant (Figure 7C, Table 1). Login to comment 265 ABCA4 p.Leu2027Phe Thus, there is a significant difference between the wild-type NBD2 protein and the L2027F mutant. Login to comment 267 ABCA4 p.Asp2177Asn ABCA4 p.Leu1971Arg ABCA4 p.Arg2038Trp ABCA4 p.Leu2027Phe ABCA4 p.Gly2146Asp ABCA4 p.Lys2175Ala Stern Volmer plots of the (A) wild-type NBD2, (B) L1971R mutant, (C) L2027F mutant, (D) D2177N mutant, (E) R2038W mutant, (F) G2146D mutant, and (F) K2175A mutant. Login to comment 273 ABCA4 p.Asp2177Asn The D2177N mutant was described earlier to have elevated levels of ATPase activity. Login to comment 281 ABCA4 p.Asp2177Asn The data were consistent with the rate of ATP hydrolysis of D2177N being increased relative to that of the wild-type protein. Login to comment 282 ABCA4 p.Arg2038Trp ABCA4 p.Gly2146Asp The R2038W and G2146D quenching profiles (Figure 7E,F and Table 1) had notable differences when compared to that of the wild-type protein. Login to comment 284 ABCA4 p.Gly2146Asp In the case of G2146D, the maximum percent quenching observed was ATP bound, 23%, ADP 13%, nucleotide-free 15%. Login to comment 286 ABCA4 p.Arg2038Trp For R2038W, the percent quenching was 30% for ATP bound versus 13% and 11% for the ADP and nucleotide-free forms. Login to comment 290 ABCA4 p.Arg2038Trp ABCA4 p.Gly2146Asp Clinically, the alteration of pattern of conformational change correlated well with the disease severity, since the mutations G2146D and R2038W are associated with STDG1 and CRD, which have an earlier age of onset and/or more rapid progression of disease. Login to comment 291 ABCA4 p.Asp2177Asn ABCA4 p.Lys2175Ala The K2175A mutant was created to explore the charge interaction between this amino acid and neighboring amino acid D2177N. Login to comment 292 ABCA4 p.Lys2175Ala The mutation K2175A led to the complete elimination of ATPase activity, however, the protein still maintained its ability to bind ATP and inferred from anisotropy data. Login to comment 295 ABCA4 p.Lys2175Ala This may likely be the underlying cause in the defect in ATP hydrolysis, since our anisotropy studies indicate that K2175A bound ATP with affinity closer to the wild type, and hence was not defective in ATP binding. Login to comment 303 ABCA4 p.Leu1971Arg ABCA4 p.Arg2038Trp ABCA4 p.Gly2146Asp The mutation L1971R has been identified with individuals suffering from the milder form of macular degeneration, Fundus Flavimaculatus, while G2146D and R2038W are associated with STGD1 and CRD, both of which are more severe forms of degeneration. Login to comment 304 ABCA4 p.Asp2177Asn Mutation D2177N has been reported in individuals suffering AMD, which is late in onset and affects the elderly. Login to comment 305 ABCA4 p.Leu1971Arg ABCA4 p.Arg2038Trp ABCA4 p.Gly2146Asp In this study, the mutations R2038W, L1971R and G2146D led to comparable (~50%) decreases in ATP hydrolysis relative to the wild-type control. Login to comment 307 ABCA4 p.Asp2177Asn Mutation D2177N led to a small increase in the rate of ATP hydrolysis accompanied by a small decrease in ATP binding affinity, as compared to the wild-type control. Login to comment 334 ABCA4 p.Asp2177Asn In the mutant, D2177N, the tryptophan residues in the NBD2‚ADP bound form were less accessible to the quencher, and only 16% quenching was observed as compared to 22% observed for wild-type NBD2‚ADP (Figure 7D, Table 1). Login to comment 336 ABCA4 p.Asp2177Asn In this mutant, the extent of quenching observed for NBD2-free was 27%, and was between that observed for NBD2‚ATP (37%) and NBD2‚ ADP (16%); hence, the general conformational pattern in D2177N was NBD2‚ATP(open) f NBD2(intermediate) f NBD2‚ ADP(taut). Login to comment 337 ABCA4 p.Asp2177Asn ABCA4 p.Asp2177Asn These data indicated that the relative magnitude of change, in going from open to taut was greater for the D2177N mutant; following ATP hydrolysis, the D2177N mutant protein assumed a more closed conformation than the wild-type protein. Login to comment 338 ABCA4 p.Asp2177Asn The D2177N mutant had an increased rate of hydrolysis that may be related to a transition to a more taut conformation upon hydrolysis of ATP to ADP. Login to comment 342 ABCA4 p.Lys2175Ala In the synthetic mutant, K2175A, the mutant protein was not observed to undergo any conformational change in response to ATP or ADP binding, as determined by the fluorescence quenching analysis (Figure 8, Table 1). Login to comment 343 ABCA4 p.Lys2175Ala Although it was able to bind ATP, mutant K2175A was completely defective in ATP hydrolysis. Login to comment 345 ABCA4 p.Lys2175Ala The K2175A mutation leads to a significant disruption of the overall charge balance of the local environment, and would result in a net-negative charge in the region due to the presence of D2177 and D2176. Login to comment 347 ABCA4 p.Leu1971Arg In the case of L1971R, dramatic changes in the quenching profiles between the wild type and mutant protein were not observed. Login to comment 348 ABCA4 p.Leu1971Arg The rate of ATP hydrolysis for L1971R was 62% of that observed for NBD2 wild type. Login to comment 352 ABCA4 p.Leu1971Arg Although L1971R was associated with a 62% decrease in ATP hydrolysis as compared to the wild type control, it was not significantly affected in terms of its structural response to nucleotide binding. Login to comment 353 ABCA4 p.Arg2038Trp ABCA4 p.Gly2146Asp The mutants R2038W and G2146D had comparable differences in nucleotide hydrolysis and thermodynamics of ATP binding. Login to comment 354 ABCA4 p.Gly2146Asp The G2146D had more closed conformations in free and nucleotide bound states; the extent of quenching for NBD2‚ATP was only 23%; the nucleotide-free and ADP-bound forms were nearly equivalent, 12% and 15%, respectively, reflecting a more closed conformation than that of wild type. Login to comment 355 ABCA4 p.Arg2038Trp The R2038W mutant was conformationaly similar the wild-type protein as evidenced in the diminished quenching of tryptophan fluorescence (Table 1). Login to comment 357 ABCA4 p.Leu2027Phe The L2027F mutant, with very low ATPase activity as demonstrated earlier, had a dramatically altered quenching profile (Table 1). Login to comment