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^Mtw1cand Ndc80c. We show that α-helical motifs in the C-termini of the Mis12c^Mtw1csubunits Dsn1, Mis12^Mtw1and Pmf1^Nnf1bind Knl1c and Ndc80c. At the opposite end of the Mis12c^Mtw1cstalk, 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^Mif2and CENP-U^Ame1, reducing their affinity for Mis12c^Mtw1. Our structure reveals how Aurora B^Ipl1phosphorylation of Dsn1^AIwould release this auto-inhibition to substantially strengthen pre-existing connections between the inner and outer kinetochore.