Investigation of the evolutionary relationship between Rio2 kinases and the canonical eukaryotic protein kinases using x‐ray crystallography

Abstract

Rio proteins are serine kinases that contain few of the conserved residues of typical protein kinases and are thus categorized as atypical protein kinases. Rio proteins are required for the cleavage of 20S rRNA to 18S rRNA.
 Canonical protein kinases are very similar to Rio2 proteins but are more active in ATPase and atuophosphorylation activity and they are onlyobserved in archaea. Computational studies have revealed that the arginine residue at position 228 highly affects the activity of canonical protein kinases. This residue in Rio family is replaced by either alanine or glycine. We were interested to study mutation at 228 of Rio2, and have found out how this mutation would affect the structure and activity of the Rio2 family. We have investigated the evolutionary relationship between Rio kinases and the canonical eukaryotic protein kinases. Our observation is that canonical eukaryotic protein kinases have evolved from Rio kinases. We have made several mutations in a key differentiating position of the kinase domain sequence of Ct‐ Rio2, G228, and have collected biochemical data to test the ability of the mutated Rio kinase to autophosphorylate and carry out ATP hydrolysis. We have also co‐crystallized Ct‐Rio2 G228 point mutants with ATP in order to determine the structural differences between the mutant and wild‐type Hence, wild type Ct‐Rio2 and five mutants of Ct‐Rio2 were purified and co‐crystalized with ATP/Mg2+. These mutants include G228A, G228D, G228N, G228R, and G228V and x‐ray diffraction data sets were collected.