Inositol Hexakisphosphate Is Bound in the ADAR2 Core and Required for RNA Editing

Abstract

We report the crystal structure of the catalytic domain of human ADAR2, an RNA editing enzyme, at 1.7 angstrom resolution. The structure reveals a zinc ion in the active site and suggests how the substrate adenosine is recognized. Unexpectedly, inositol hexakisphosphate (IP 6 ) is buried within the enzyme core, contributing to the protein fold. Although there are no reports that adenosine deaminases that act on RNA (ADARs) require a cofactor, we show that IP 6 is required for activity. Amino acids that coordinate IP 6 in the crystal structure are conserved in some adenosine deaminases that act on transfer RNA (tRNA) (ADATs), related enzymes that edit tRNA. Indeed, IP 6 is also essential for in vivo and in vitro deamination of adenosine 37 of tRNA ala by ADAT1.One form of RNA editing is catalyzed by adenosine deaminases that act on RNA (ADARs), a family of enzymes that deaminate adenosine to form inosine in double-stranded RNA (dsRNA) (Fig. 1A) (1). ADARs are important for proper neuronal function (2-4) and also are implicated in the regulation of RNA interference (RNAi) (5-7). Inosine is recognized as guanosine by most cellular proteins and the translation machinery, and it pairs most stably with cytidine. Therefore, editing of RNA can alter a codon, create splice sites, and change its structure. The latter occurs when an AU base pair is changed to an IU mismatch and may be important for the effects of ADARs on the RNAi pathway.ADARs from all organisms have a common domain structure consisting of one to three dsRNA binding motifs (dsRBMs) near the N terminus, followed by a conserved C-terminal catalytic domain (1,8). Human ADAR2 (hADAR2) contains two dsRBMs, and its best characterized substrates are the pre-mRNAs of glutamate and serotonin receptors (9,10). Editing of codons within these RNAs leads to altered amino acids and generates receptors with altered function. hADAR2 also edits its own message to create a new splice site (11). Purified hADAR2 deaminates substrates in vitro (12) in the absence of any added cofactors, and deletions of Nterminal sequences, including dsRBM1, result in an active protein that accurately edits an RNA substrate (13). In addition, we found that a protein consisting of only the catalytic deaminase domain of hADAR2 (hADAR2-D, residues 299 to 701) ( fig. S1A) was active in vitro, although it deaminates RNA less efficiently than full-length hADAR2 ( fig. S1B).

Editor meets silencer: crosstalk between RNA editing and RNA interference

“…The C-terminal region of ADAR contains amino-acid residues that are conserved in several cytidine deaminases and are predicted to participate in the formation of the catalytic centre of ADAR 30,38 . The crystal structure of the catalytic domain of human ADAR2 shows that His394, Glu396 and two Cys residues, Cys451 and Cys516, of ADAR2 are indeed involved in the coordination of a zinc atom and the formation of the catalytic centre 39 . Most surprisingly, however, the structural studies also revealed the presence of inositol hexakisphosphate (IP 6 ) buried in the enzyme core, but located very close to the catalytic centre.…”

“…Most surprisingly, however, the structural studies also revealed the presence of inositol hexakisphosphate (IP 6 ) buried in the enzyme core, but located very close to the catalytic centre. The IP 6 molecule could have a crucial role during the deamination reaction 39 .…”

Large-Scale Overexpression and Purification of ADARs from Saccharomyces cerevisiae for Biophysical and Biochemical Studies

“…The RNA-binding domain consists of one to three double-stranded RNA-binding motifs (dsRBM). The catalytic domain consists of a deaminase motif that resembles Escherichia coli cytidine deaminase and binds zinc, as well as a motif that coordinates the small molecule myo-inositol hexakisphosphate (IP 6 , Maas et al, 2003;Macbeth et al, 2005). In addition, human and Xenopus ADAR1 contain two Z-DNA-binding motifs N-terminal to their three dsRBMs (Maas et al, 2003).…”

Substrate-dependent Contribution of Double-stranded RNA-binding Motifs to ADAR2 Function

“…ADAR2 displays a modular organization with two tandem dsRNA-binding motifs (dsRBMs) connected by a flexible linker and a conserved adenosine deaminase domain toward the carboxy terminus, for which the structures have recently been determined (Macbeth et al, 2005;Stefl et al, 2006). The dsRBM is a highly conserved 65-75 amino acid (aa) domain, present in many eukaryotic proteins with diverse cellular functions, that forms an ␣1-␤1-␤2-␤3-␣2 topology in which the two ␣-helices are packed along a face of a three-stranded antiparallel ␤-sheet, with most of the potential RNA-binding residues exposed on one surface (FierroMonti and Mathews, 2000;Tian et al, 2004).…”

“…The most extensively studied substrates of Ato-I conversion are transcripts encoding ionotropic glutamate receptor (GluR) subunits and the 2C-subtype of the serotonin receptor (5-HT 2C R), in which editing leads to nonsynonymous codon changes that generate channels with altered electrophysiologic and ion permeation properties (Seeburg and Hartner, 2003) and receptors with decreased G protein-coupling efficiency (Burns et al, 1997;Niswender et al, 1999;Berg et al, 2001). A-to-I modifications have also been described in nontranslated RNAs and noncoding regions of mRNA transcripts, suggesting that such RNA modifications affect other aspects of RNA function, including splicing, translation efficiency, nuclear retention, and transcript stability (Rueter et al, 1999;Athanasiadis et al, 2004;Blow et al, 2004;Kim et al, 2004;Levanon et al, 2004;DeCerbo and Carmichael, 2005;Prasanth et al, 2005).ADAR2 displays a modular organization with two tandem dsRNA-binding motifs (dsRBMs) connected by a flexible linker and a conserved adenosine deaminase domain toward the carboxy terminus, for which the structures have recently been determined (Macbeth et al, 2005;Stefl et al, 2006). The dsRBM is a highly conserved 65-75 amino acid (aa) domain, present in many eukaryotic proteins with diverse cellular functions, that forms an ␣1-␤1-␤2-␤3-␣2 topology in which the two ␣-helices are packed along a face of a three-stranded antiparallel ␤-sheet, with most of the potential RNA-binding residues exposed on one surface (FierroMonti and Mathews, 2000;Tian et al, 2004).…”