ABCMdb

PMID: 20404325

Biswas-Fiss EE, Kurpad DS, Joshi K, Biswas SB
Interaction of extracellular domain 2 of the human retina-specific ATP-binding cassette transporter (ABCA4) with all-trans-retinal.
J Biol Chem. 2010 Jun 18;285(25):19372-83. Epub 2010 Apr 19., [PubMed]

Sentences

No. Mutations Sentence Comment

112 ABCA4 p.Cys1488Arg ABCA4 p.Arg1443His ABCA4 p.Trp1408Leu Three ABCA4 mutations associated with Stargardt disease, W1408L, R1443H, and C1488R, were chosen for analysis in this study. Login to comment 133 ABCA4 p.Cys1488Arg ABCA4 p.Arg1443His ABCA4 p.Trp1408Leu We have analyzed changes in the conformation of ECD2 protein harboring disease-associated mutations W1408L, C1488R, and R1443H. Login to comment 135 ABCA4 p.Trp1408Leu ABCA4 p.Trp1408Leu W1408L Mutation-The W1408L mutation has been reported in several studies investigating the ABCA4 genotype of individuals afflicted with Stargardt disease, fundus flavimaculatus, and cone rod dystrophy (13, 19, 48). Login to comment 144 ABCA4 p.Trp1408Leu The thermal melting profile of the W1408L is shown in Fig. 6B. Login to comment 160 ABCA4 p.Cys1488Arg ABCA4 p.Arg1443His ABCA4 p.Trp1408Leu TABLE 1 Stargardt disease-associated mutations occurring in the ECD2 domain investigated in this study Mutation Base change Mutagenesis primer R1443H CGC4328CAC 5b18;-CAGGCTTTGGCAACCACTGAAGGAAGGGTGGCTTC-3b18; W1408L TGG4221TTG 5b18;-ACCCTTCACCCCTTGATATGGGCAGCAGTACACCTTC-3b18; C1488R TGC4462CGC 5b18;-AACCCTTCACCATCCCGCAGGTGCAGCACCAGGGAGAAG-3b18; Second Extracellular Domain of ABCA4 19376 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 285ߦNUMBER 25ߦJUNE 18, 2010 wtECD2, indicated by a Tm of 57.1 &#b0;C (Table 3). Login to comment 161 ABCA4 p.Cys1488Arg ABCA4 p.Arg1443His ABCA4 p.Trp1408Leu TABLE 1 Stargardt disease-associated mutations occurring in the ECD2 domain investigated in this study Mutation Base change Mutagenesis primer R1443H CGC4328CAC 5Ј-CAGGCTTTGGCAACCACTGAAGGAAGGGTGGCTTC-3Ј W1408L TGG4221TTG 5Ј-ACCCTTCACCCCTTGATATGGGCAGCAGTACACCTTC-3Ј C1488R TGC4462CGC 5Ј-AACCCTTCACCATCCCGCAGGTGCAGCACCAGGGAGAAG-3Ј Second Extracellular Domain of ABCA4 19376 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 285•NUMBER 25•JUNE 18, wtECD2, indicated by a Tm of 57.1 °C (Table 3). Login to comment 162 ABCA4 p.Cys1488Arg ABCA4 p.Cys1488Arg C1488R Mutation-The C1488R mutation has been identified as a Stargardt disease-linked mutation in individuals of Northern and Central European ancestry (13, 23, 47). Login to comment 163 ABCA4 p.Gly863Ala ABCA4 p.Cys1488Arg ABCA4 p.Cys1488Arg C1488R Mutation-The C1488R mutation has been identified as a Stargardt disease-linked mutation in individuals of Northern and Central European ancestry (13, 23, 47). Login to comment 164 ABCA4 p.Gly863Ala ABCA4 p.Cys1488Arg This mutation has been reported to occur in the ABCA4 gene as an individual heterozygous mutation as well as in trans with G863A mutation. This mutation represents a chemically significant substitution of a basic residue for a strictly conserved cysteine residue. Login to comment 165 ABCA4 p.Cys1488Arg The far-UV CD spectrum of the C1488R mutant at 25 °C is presented in Fig. 5. Login to comment 170 ABCA4 p.Arg1443His ABCA4 p.Arg1443His R1443H Mutation-The R1443H mutation has been identified in individuals with Stargardt disease and appears to occur in those with Northern European as well as East Indian ancestry (23, 51). Login to comment 171 ABCA4 p.Arg1443His ABCA4 p.Arg1443His R1443H Mutation-The R1443H mutation has been identified in individuals with Stargardt disease and appears to occur in those with Northern European as well as East Indian ancestry (23, 51). Login to comment 173 ABCA4 p.Arg1443His Major shifts in the CD spectral minima at 208 and 220 nm for R1443H were observed at 25 &#b0;C, consistent with a helix-coil transition concomitant with a significant change of overall secondary structure (Fig. 5). Login to comment 174 ABCA4 p.Arg1443His Major shifts in the CD spectral minima at 208 and 220 nm for R1443H were observed at 25 °C, consistent with a helix-coil transition concomitant with a significant change of overall secondary structure (Fig. 5). Login to comment 175 ABCA4 p.Arg1443His Comparing the spectra of the native and heat-denatured states of the R1443H mutant, it appears that the heat-induced alterations of the secondary structure elements over the temperature range of 20-80 &#b0;C were small. Login to comment 176 ABCA4 p.Arg1443His ABCA4 p.Arg1443His Comparing the spectra of the native and heat-denatured states of the R1443H mutant, it appears that the heat-induced alterations of the secondary structure elements over the temperature range of 20-80 °C were small. Login to comment 177 ABCA4 p.Arg1443His It is likely that the absence of a sequential loss of structure with temperature may simply reflect the lack of ␣-helical structure of R1443H relative to wtECD2. Login to comment 204 ABCA4 p.Cys1488Arg ABCA4 p.Arg1443His ABCA4 p.Trp1408Leu A, wild type ECD2 ; inset, ellipticity at 220 nm was monitored over the range of 20-80 &#b0;C, and the Tm was determined from the inflection points of data fitted to sigmoidal curve; B, W1408L; C, C1488R, and D, R1443H. Login to comment 205 ABCA4 p.Cys1488Arg ABCA4 p.Cys1488Arg ABCA4 p.Arg1443His ABCA4 p.Trp1408Leu ABCA4 p.Trp1408Leu A, wild type ECD2 ; inset, ellipticity at 220 nm was monitored over the range of 20-80 °C, and the Tm was determined from the inflection points of data fitted to sigmoidal curve; B, W1408L; C, C1488R, and D, R1443H. Login to comment 206 ABCA4 p.Cys1488Arg ABCA4 p.Trp1408Leu TABLE 3 Summary of secondary structure analysis and binding constants for the ABCA4-ECD2 WT and mutant polypeptides Genotype ␣-Helix beta-Sheet Random coil Tm Kd, app °C M Wild type 0.25 0.20 0.55 49.7 1.7 Ϯ 1.1 ϫ 10-7 R1433H 0.11 0.39 0.50 NDa ND W1408L 0.23 0.24 0.53 57.1 8.1 Ϯ 1.9 ϫ 10-7 C1488R 0.20 0.27 0.53 66.7 6.7 Ϯ 1.4 ϫ 10-7 a ND means not determined. Login to comment 208 ABCA4 p.Cys1488Arg ABCA4 p.Trp1408Leu With both W1408L and C1488R, attenuation of tryptophan fluorescence emission was observed with increasing concentrations of all-trans-retinal (Fig. 7, B and C), albeit significantly diminished compared with that observed with the wild type ECD2 (Fig. 7A). Login to comment 209 ABCA4 p.Cys1488Arg ABCA4 p.Arg1443His ABCA4 p.Trp1408Leu With both W1408L and C1488R, attenuation of tryptophan fluorescence emission was observed with increasing concentrations of all-trans-retinal (Fig. 7, B and C), albeit significantly diminished compared with that observed with the wild type ECD2 (Fig. 7A). Login to comment 210 ABCA4 p.Arg1443His On the other hand, mutant R1443H displayed little change in tryptophan fluorescence spectra in the presence FIGURE 7. Login to comment 216 ABCA4 p.Cys1488Arg ABCA4 p.Arg1443His ABCA4 p.Trp1408Leu લ, ECD2 WT; , R1443H; ǧa;, W1408L; ƪb;, C1488R. Login to comment 217 ABCA4 p.Cys1488Arg ABCA4 p.Arg1443His ABCA4 p.Trp1408Leu ✸, ECD2 WT; , R1443H; Ⅺ, W1408L; ᭛, C1488R. Login to comment 223 ABCA4 p.Cys1488Arg ABCA4 p.Trp1408Leu The Kd,app values for W1408L and C1488R were (8.1 afe; 1.9) afb; 10afa;7 and (6.7 afe; 1.4) afb; 10afa;7 M, respectively. Login to comment 224 ABCA4 p.Cys1488Arg ABCA4 p.Trp1408Leu The Kd,app values for W1408L and C1488R were (8.1 Ϯ 1.9) ϫ 10-7 and (6.7 Ϯ 1.4) ϫ 10-7 M, respectively. Login to comment 250 ABCA4 p.Arg1443His Influence of Stargardt Disease-associated Mutations on ECD2 Domain Secondary Structure-Of the three mutants examined here, the R1443H mutation appeared to be the most severe in FIGURE 10. Login to comment 251 ABCA4 p.Arg1443His Influence of Stargardt Disease-associated Mutations on ECD2 Domain Secondary Structure-Of the three mutants examined here, the R1443H mutation appeared to be the most severe in FIGURE 10. Login to comment 262 ABCA4 p.Trp1408Leu A minimal change in the CD spectrum was observed with the W1408L mutation. This mutant appeared to have secondary structural parameters that were comparable with that of the wild type ECD2 polypeptide. Login to comment 263 ABCA4 p.Trp1408Leu A minimal change in the CD spectrum was observed with the W1408L mutation. This mutant appeared to have secondary structural parameters that were comparable with that of the wild type ECD2 polypeptide. Login to comment 265 ABCA4 p.Cys1488Arg The C1488R mutation appeared to be intermediate in nature with respect to its influence on the ECD2 structure. Login to comment 266 ABCA4 p.Cys1488Arg The C1488R mutation appeared to be intermediate in nature with respect to its influence on the ECD2 structure. Login to comment 296 ABCA4 p.Arg1443His In the case of mutant R1443H, a large decrease in the binding affinity was observed, with a b0e;250-fold increase in Kd (Fig. 8 and Table 3). Login to comment 297 ABCA4 p.Cys1488Arg ABCA4 p.Arg1443His ABCA4 p.Trp1408Leu In the case of mutant R1443H, a large decrease in the binding affinity was observed, with a Ͼ250-fold increase in Kd (Fig. 8 and Table 3). Login to comment 298 ABCA4 p.Cys1488Arg ABCA4 p.Arg1443His ABCA4 p.Trp1408Leu Less dramatic changes were seen with the other mutants investigated in this study, with a 5-8-fold increase in Kd for W1408L and C1488R, respectively. Login to comment 299 ABCA4 p.Arg1443His With mutant R1443H, the apparent lack of interaction with all-trans-retinal was observed. Login to comment 301 ABCA4 p.Arg1443His The conformation of R1443H was strik- Second Extracellular Domain of ABCA4 JUNE 18, 2010ߦVOLUME 285ߦNUMBER 25 JOURNAL OF BIOLOGICAL CHEMISTRY 19381 at SEMMELWEIS UNIV OF MEDICINE on December , ingly different from that of wtECD2 and appeared refractory to changes in temperature. Login to comment 302 ABCA4 p.Arg1443His The conformation of R1443H was strik- Second Extracellular Domain of ABCA4 JUNE 18, 2010•VOLUME 285•NUMBER JOURNAL OF BIOLOGICAL CHEMISTRY 19381 ingly different from that of wtECD2 and appeared refractory to changes in temperature. Login to comment 303 ABCA4 p.Cys1488Arg ABCA4 p.Trp1408Leu Likewise, the relatively moderate structural changes of W1408L and C1488R also correlated with more moderate changes in interaction with all-trans-retinal as compared with the wild type ECD2. Login to comment 304 ABCA4 p.Cys1488Arg ABCA4 p.Trp1408Leu Likewise, the relatively moderate structural changes of W1408L and C1488R also correlated with more moderate changes in interaction with all-trans-retinal as compared with the wild type ECD2. Login to comment