ABCMdb

ABCA4 p.Lys2175Ala

Predicted by SNAP2:	A: N (53%), C: N (53%), D: N (57%), E: N (61%), F: D (53%), G: N (57%), H: N (66%), I: N (61%), L: N (57%), M: N (57%), N: N (78%), P: N (57%), Q: N (72%), R: N (87%), S: N (72%), T: N (78%), V: N (57%), W: D (66%), Y: N (57%),
Predicted by PROVEAN:	A: N, C: D, D: N, E: N, F: D, G: N, H: N, I: D, L: D, M: N, N: N, P: N, Q: N, R: N, S: N, T: N, V: D, W: D, Y: D,

[switch to compact view]
Comments [show]

None has been submitted yet.

Publications
[hide] Functional analysis of genetic mutations in nucleo... Biochemistry. 2003 Sep 16;42(36):10683-96. 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., [PMID:12962493]

Abstract [show]
The rod outer segment (ROS) ABC transporter (ABCR) plays an important role in the outer segment of retinal rod cells, where it functions as a transporter of all-trans retinal, most probably as the complex lipid, retinylidene-phosphatidyl-ethanolamine. We report here a quantitative analysis of the structural and functional effects of genetic mutations, associated with several macular degenerations, in the second nucleotide-binding domain of ABCR (NBD2). We have analyzed the ATP binding, kinetics of ATP hydrolysis, and structural changes. The results of these multifaceted analyses were correlated with the disease severity and prognosis. Results presented here demonstrated that, in wild type NBD2, distinct conformational changes accompany nucleotide (ATP and ADP) binding. Upon ATP binding, NBD2 protein changed to a relaxed conformation where tryptophans became more solvent-exposed, while ADP binding reverses this process and leads back to a taut conformation that is also observed with the unbound protein. This sequence of conformational change appears to be important in the energetics of the ATP hydrolysis and may have important structural consequences in the ability of the NBD2 domain to act as a regulator of the nucleotide-binding domain 1. Some of the mutant proteins displayed strikingly different patterns of conformational changes upon nucleotide binding that pointed to unique structural consequences of these genetic mutations. The ABCR dysfunctions, associated with various retinopathies, are multifaceted in nature and include alterations in protein structure as well as the attenuation of ATPase activity and nucleotide binding.

Comments [show]

None has been submitted yet.

Sentences [show]
No. Sentence Comment
73 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 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
144 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 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 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
188 We created a site-specific mutant, K2175A, to explore this hypothesis. Login to comment
189 K2175A is not a naturally occurring disease-associated mutation. Login to comment
190 Analyses of the putative salt-bridge mutant, K2175A, demonstrated that this change led to the loss of ATPase activity. Login to comment
191 The lack of detectable levels of ATPase activity precluded kinetic analysis of the K2175A mutant. Login to comment
192 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 The ATP binding constants for D2177N and K2175A were determined similarly (Figures 6B,E). Login to comment
220 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 In both of these mutants, particularly in the case of K2175A, alteration in ATP binding was minor. Login to comment
224 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
253 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
267 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
291 The K2175A mutant was created to explore the charge interaction between this amino acid and neighboring amino acid D2177N. Login to comment
292 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 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
342 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 Although it was able to bind ATP, mutant K2175A was completely defective in ATP hydrolysis. Login to comment
345 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