The C‐terminus of VRK1 Governs Interactions with Nucleosomes to Influence Histone H3 Threonine 3 (H3T3) Phosphorylation

Vaccinia‐related kinase 1 (VRK1) is a highly abundant nuclear kinase that has numerous functions during mitosis, including the phosphorylation of the histone H3 tail at threonine 3 (H3T3ph). This post‐translational modification, attributed to both VRK1 and the haspin kinase, is crucial for ensuring appropriate chromosome segregation. Despite the importance of VRK1 to cell cycle regulation, little is known about the biochemical basis of its activity on nucleosomes. Because placement of histone phosphorylation during mitosis is tightly spatiotemporally regulated, we sought to determine the chromatin template itself serves as a regulatory mechanism for VRK1 activity. In this work, we characterize the interaction between VRK1 and nucleosomes and determine how nucleosome binding affects nucleosome‐directed kinase activity at H3T3. We find that VRK1 directly and stably interacts with nucleosomes with an apparent K d of ~200 nM. VRK1 binds nucleosomes via a C‐terminal alternating arginine motif, mutations or truncations of which are found in rare cases of genetic spinal muscular atrophy. Mutation or truncation of this motif leads to a 4‐50 fold reduction in nucleosome affinity that is correlated with mutation position and severity. C‐terminal arginine mutants of VRK1 show reduced capacity for histone H3T3 phosphorylation, suggesting that stable interaction with nucleosomes is required for nucleosome‐directed kinase activity at H3T3. The nucleosome acidic patch, a negatively charged region at the dimer interface of H2A/H2B, is a hot spot for arginine‐mediated nucleosome interactions. Neutralization of the acidic patch impairs VRK1 binding but surprisingly increases the activity of VRK1 at H3T3. In contrast, the presence of extended lengths of linker DNA promotes both nucleosome binding and phosphorylation of H3T3. Cryo‐EM single particle analysis of VRK1‐bound nucleosome particles demonstrates both acidic patch binding and heterogenous DNA binding that concentrates around the nucleosome linker DNA. This evidence suggests that VRK1 adopts at least two alternative binding modes on the nucleosome (acidic patch‐governed vs. linker DNA‐governed) that either promote or inhibit nucleosome‐directed kinase activity based on the position and distance of VRK1 from the H3 tail. We suggest a model wherein dynamic access to the nucleosome acidic patch regulates the activity of VRK1 towards H3T3 during mitosis.