Adenosine Deaminases Acting on RNA (ADARs) are a family of enzymes that catalyze the hydrolytic deamination of Adenosine (A) to Inosine (I). Since Inosine is recognized as Guanosine during translation, A-to-I editing can change the meaning of codons, introduce or remove splice sites, or affect protein functions and structures. In human, there are two catalytically active form of ADARs, ADAR1 and ADAR2. Many studies have shown that aberrant activities of ADARs are highly associated with various human disease including cancers. In 2016, our lab in collaboration with Dr. Andrew Fisher’s lab at UC Davis, solved a crystal structure of human ADAR2 catalytic domain (hADAR2d) bound to double stranded RNA (dsRNA) using an adenosine analog, 8-azanebularine to stabilize the complex. This crystal structure provided insights into substrate recognition by hADAR2d as well as possible explanations for its specificity. However, 8-azanebularine trapping is only useful for a ADAR-dsRNA complex bearing a tetrahedral intermediate mimic because 8-azanebularine is hydrated by ADAR and trapped as tetrahedral intermediate mimic in the active site. Therefore, we attempted to stabilize a protein-RNA complex using a covalent trapping method called disulfide crosslinking, which utilizes a disulfide bond between a cysteine residue of mutant protein and thiol modified dsRNA as a strategy to obtain stabilized ADAR-dsRNA complexes. Disulfide crosslinking of both ADAR1 and a ADAR2 catalytic domain and dsRNA containing a thiol modification clearly showed the formation of crosslinked complexes. However, a background crosslinking was observed because native Cys residues within a catalytic domain of both ADAR1 and ADAR2 could interact with a thiol modified dsRNA. To remove the background crosslinking, library was generated for each native Cys residue to find out the optimal amino acid to mutate the native Cys residue to maximize a specific crosslinking with a mutated Cys residue of mutant protein. Overall, disulfide crosslinking results show that it is a promising trapping strategy which allow us to use it for solving an x-ray crystal structure of various ADAR-dsRNA complexes.

Covalent trapping of human ADAR catalytic domain using thiol modified dsRNA

256th National Meeting (2018)

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