Abstract 1711 VRK1 Mutants and Implications for Kinase Stability and Activity in Neurodegenerative Disorders

Vaccinia-related kinase 1 (VRK1) is a serine/threonine kinase that contributes to the regulation of mitosis, the DNA damage response, and other processes. Point mutations within the VRK1 sequence are associated with a variety of rare complex neurodegenerative disorders. Previous research suggests this may be due to changes in kinase activity and/or structure and stability, leading to downstream defects in Cajal body assembly. The D263G variant was identified in patients with hereditary spastic paraplegia. The missense mutation R321C has been detected in patients showing probable signs of ALS including lower extremity weakness and motor neuron lesions. Both mutants are on the surface of the C-terminal lobe of the kinase. This study focuses on understanding the impact of these mutations on protein stability and ligand binding affinity. The mutations were produced in a His-tagged VRK1 construct plasmid which was then transformed into E. coli, allowing for the overexpression and purification of the specific mutated VRK1 proteins. The stability and secondary structure of the protein were analyzed using circular dichroism (CD), and the protein's ligand binding ability was evaluated using differential scanning fluorimetry (DSF) with both nucleotides and kinase inhibitors. Results were compared to the wild type VRK1 protein and results for previously studied VRK1 variants.