Assembly and phospho-regulatory mechanisms of the budding yeast outer kinetochore KMN complex

During mitosis and meiosis kinetochores mediate interactions between chromosomes and spindle microtubules. Kinetochores are multi-megadalton protein complexes essential for chromosome segregation in all eukaryotes, however recent structural, functional, and evolutionary studies have revealed divergent mechanisms of kinetochore assembly. In this study, we use cryo-EM to understand the structural mechanisms by which the budding yeast microtubule-binding outer kinetochore KMN complex assembles, and how its interactions with the centromere-binding inner kinetochore are regulated. The ten-subunit KMN complex comprises three subcomplexes: Knl1c, Mis12c ^Mtw1c and Ndc80c. We show that α-helical motifs in the C-termini of the Mis12c ^Mtw1c subunits Dsn1, Mis12 ^Mtw1 and Pmf1 ^Nnf1 bind Knl1c and Ndc80c. At the opposite end of the Mis12c ^Mtw1c stalk, an N-terminal auto-inhibitory segment of Dsn1 (Dsn1 ^AI ) folds into two α-helices that engage its head 1 domain, thereby occluding binding sites for the inner kinetochore subunits CENP-C ^Mif2 and CENP-U ^Ame1 , reducing their affinity for Mis12c ^Mtw1 . Our structure reveals how Aurora B ^Ipl1 phosphorylation of Dsn1 ^AI would release this auto-inhibition to substantially strengthen pre-existing connections between the inner and outer kinetochore.