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

Crosslinking of KMN outer kinetochore 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, Mis12cMtw1c and Ndc80c. We show that α-helical motifs in the C-termini of the Mis12cMtw1c subunits Dsn1, Mis12Mtw1 and Pmf1Nnf1 bind Knl1c and Ndc80c. At the opposite end of the Mis12cMtw1c stalk, an N-terminal auto-inhibitory segment of Dsn1 (Dsn1AI) folds into two α-helices that engage its head 1 domain, thereby occluding binding sites for the inner kinetochore subunits CENP-CMif2 and CENP-UAme1, reducing their affinity for Mis12cMtw1. Our structure reveals how Aurora BIpl1 phosphorylation of Dsn1AI would release this auto-inhibition to substantially strengthen pre-existing connections between the inner and outer kinetochore.