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bioRxiv · 10.1101/2025.06.03.657598

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

Turner, N. N.·Zhang, Z.·Muir, K. W.·McLaughlin, S. H.·Morgan, T.·Barford, D.
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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, Mis12cMtw1c and Ndc80c. We show that -helical motifs in the C-termini of the Mis12cMtw1c subunits Dsn1, Mis12Mtw1 and Nnf1 bind Knl1c and Ndc80c. At the opposite end of the Mis12cMtw1c stalk, an N-terminal auto-inhibitory segment of Dsn1 (Dsn1AI) folds into two -helices that engage the Mis12cMtw1c 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. SummaryKinetochore-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.

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Turner, N. N., Zhang, Z., Muir, K. W., McLaughlin, S. H., Morgan, T., Barford, D.. 2025-06-04. Assembly and phospho-regulatory mechanisms of the budding yeast outer kinetochore KMN complex. https://doi.org/10.1101/2025.06.03.657598

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