Assembly and phosphoregulatory mechanisms of the budding yeast outer kinetochore KMN complex

Abstract 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 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 the Mis12c Mtw1c 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. Summary Kinetochore-localised KMN complexes mediate chromosome segregation by attaching chromosomes to spindle microtubules. Cryo-EM, biochemical and genetic experiments reveal conserved underlying mechanisms of complex assembly, auto-inhibition and phospho-regulation that ensure precise control of KMN assembly onto centromeres to enable genome division.