ABCMdb

ABCA4 p.Thr1979Ala

ClinVar:	c.5936C>T , p.Thr1979Ile ? , not provided
Predicted by SNAP2:	A: N (57%), C: D (53%), D: N (53%), E: N (57%), F: D (66%), G: D (53%), H: N (66%), I: D (91%), K: N (61%), L: D (59%), M: D (53%), N: N (66%), P: D (53%), Q: N (57%), R: N (53%), S: N (78%), V: N (53%), W: D (80%), Y: D (63%),
Predicted by PROVEAN:	A: D, C: D, D: D, E: D, F: D, G: D, H: D, I: D, K: D, L: D, M: D, N: D, P: D, Q: D, R: D, S: D, V: D, W: D, Y: D,

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[hide] Interaction of the nucleotide binding domains and ... Biochemistry. 2006 Mar 21;45(11):3813-23. 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., [PMID:16533065]

Abstract [show]
We report here a novel regulation of the ATPase activity of the human retina specific ATP binding cassette transporter (ABC), ABCR, by nucleotide binding domain interactions. We also present evidence that recombinant nucleotide binding domains of ABCR interact in vitro in the complete absence of transmembrane domains (TMDs). Although similar domain-domain interactions have been described in other ABC transporters, the roles of such interactions on the enzymatic mechanisms of these transporters have not been demonstrated experimentally. A quantitative analysis of the in vitro interactions as a function of the nucleotide-bound state demonstrated that the interaction takes place in the absence of nucleotide as well as in the presence of ATP and that it only attenuates in the ADP-bound state. Analysis of the ATPase activities of these proteins in free and complex states indicated that the NBD1-NBD2 interaction significantly influences the ATPase activity. Further investigation, using site-specific mutants, showed that mutations in NBD2 but not NBD1 led to the alteration of the ATPase activity of the NBD1.NBD2 complex and residue Arg 2038 is critical to this regulation. These data indicate that changes in the oligomeric state of the nucleotide binding domains of ABCR are coupled to ATP hydrolysis and might represent a possible signal for the TMDs of ABCR to export the bound substrate. Furthermore, the data support a mechanistic model in which, upon binding of NBD2, NBD1 binds ATP but does not hydrolyze it or does so with a significantly reduced rate.

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74 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 Lanes (from left to right): 1, NBD1WT; 2, NBD1P940R; 3, NBD1K969A,T970A; 4, NBD2WT; 5, NBD2R2038W; 6, NBD2K1978A,T1979A. Login to comment
198 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
218 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
225 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 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