Crystal structure of the yeast heterodimeric ADAT2/3 deaminase

Abstract

Background The adenosine-to-inosine (A-to-I) editing in anticodons of tRNAs is critical for wobble base-pairing during translation. This modification is produced via deamination on A34 and catalyzed by the adenosine deaminase acting on tRNA (ADAT) enzyme. Eukaryotic ADATs are heterodimers composed of the catalytic subunit ADAT2 and the structural subunit ADAT3, but their molecular assemblies and catalytic mechanisms are largely unclear. Results Here, we report a 2.8-Å crystal structure of Saccharomyces cerevisiae ADAT2/3 (ScADAT2/3), revealing its heterodimeric assembly and substrate recognition mechanism. While each subunit clearly contains a domain resembling their prokaryotic homolog TadA, suggesting an evolutionary gene duplication event, they also display accessory domains for additional structural or functional purposes. The N-lobe of ScADAT3 exhibits a positively charged region with a potential role in the recognition and binding of tRNA, supported by our biochemical analysis. Interestingly, ScADAT3 employs its C-terminus to block tRNA’s entry into its pseudo-active site and thus inactivates itself for deamination despite the preservation of a zinc-binding site, a mechanism possibly shared only among yeasts. Conclusions Combining the structural with biochemical, bioinformatic, and in vivo functional studies, we propose a stepwise model for the pathway of deamination by ADAT2/3. Our work provides insight into the molecular mechanism of the A-to-I editing by the eukaryotic ADAT heterodimer, especially the role of ADAT3 in catalysis.

The structure of the mouse ADAT2/ADAT3 complex reveals the molecular basis for mammalian tRNA wobble adenosine-to-inosine deamination

“…While this manuscript was in revision, the yeast ADAT complex structure was published ( 46 ). Conclusions of the study on the yeast complex are similar to ours, notably on the role of ADAT3/Tad3p N-terminal domain for tRNA recognition and presentation to ADAT2/Tad2p active site, as well as the inactivation of ADAT3/Tad3p by capping of its zinc binding site.…”

Functional and structural investigation of N-terminal domain of the SpTad2/3 heterodimeric tRNA deaminase

“…We first cloned the full-length tad2 gene into both the pET-28a and the pET-21b vectors for expression tests. Both constructs were poorly expressed in E. coli , suggesting that the SpTad2 protein needs the stabilization from the accessory subunit SpTad3, as was demonstrated in the ScTad2/3 case [10] . We also removed the first eleven residues from SpTad2, which was predicted to form a disordered region, but the truncated form was not expressed either (data not shown).…”

“…In our case, N-SpTad2 was not found to show binding abilities to DNA or tRNA. In our previous studies, we consider species with Tad3s that end with two cysteine residues were more ancestral species [10] . Therefore, the Schizosaccharomyces pombe tad3 gene may occur later than its Saccharomyces cerevisiae counterpart.…”

“…Importantly, we discovered that the N-terminus of ScTad3 forms a stand-alone domain and contributes to the binding of tRNA. Moreover, ScTad3 employs its C-terminal cysteine to block the pseudo-catalytic pocket and eliminate its own deamination activity [10] . In this study, we first identified a potential nucleotide kinase located at the N-terminus of Schizosaccharomyces pombe Tad2 (N-SpTad2).…”

Inosine in Biology and Disease

“…A conserved proton-shuttling glutamate, which is essential for the catalytic activity of ADAT2, was lost during the evolution of ADAT3, rendering this subunit catalytically inactive [ 76 , 77 ]. A recent study on the crystal structure of ADAT2/3 from Saccharomyces cerevisiae suggests that the positively charged residues at the N-terminal region of ADAT3 may play a role in substrate recognition [ 78 ].…”