Unconventional kinetochore kinases KKT2 and KKT3 have a unique zinc finger that promotes their kinetochore localization

<jats:title>Abstract</jats:title><jats:p>Chromosome segregation in eukaryotes is driven by the kinetochore, a macromolecular protein complex that assembles onto centromeric DNA and binds spindle microtubules. Cells must tightly control the number and position of kinetochores so that all chromosomes assemble a single kinetochore. A central player in this process is the centromere-specific histone H3 variant CENP-A, which localizes specifically within centromeres and promotes kinetochore assembly. However, CENP-A is absent from several eukaryotic lineages including kinetoplastids, a group of evolutionarily divergent eukaryotes that have an unconventional set of kinetochore proteins. It remains unknown how kinetoplastids specify kinetochore positions or promote kinetochore assembly in the absence of CENP-A. Here we studied two homologous kinetoplastid kinases (KKT2 and KKT3) that localize constitutively at centromeres. KKT2 and KKT3 central domains were sufficient for centromere localization in <jats:italic>Trypanosoma brucei</jats:italic>. Crystal structures of the KKT2 central domain from two divergent kinetoplastids revealed a unique zinc finger domain, which promotes its kinetochore localization in <jats:italic>T. brucei</jats:italic>. Mutations in the equivalent zinc finger domain of KKT3 abolished its kinetochore localization and function. This study lays the foundation for understanding the mechanism of kinetochore specification and assembly in kinetoplastids.</jats:p>