Assembly and autoinhibitory mechanisms of the budding yeast kinetochore KMN complex

Abstract

During eukaryotic mitosis, kinetochore protein complexes form the primary and essential linkage between spindle microtubules and chromosomes. Kinetochores directly mediate chromosome segregation by forming robust attachments to microtubules under applied force, and act to ensure the accuracy of chromosome segregation by scaffolding signalling pathways that regulate cell cycle progression and kinetochore-microtubule (KT-MT) attachment strength. An inner kinetochore complex binds to centromeric chromatin and recruits the outer kinetochore KMN complex, which is composed of the Knl1c, Mtw1c, and Ndc80c subcomplexes. Ndc80c binds to the ends of spindle microtubules in a manner that is sensitive to error correction (EC) signalling pathways. The EC pathway reduces the strength of Ndc80c-mediated KT-MT attachments at chromosomes that are incorrectly attached to the spindle. Knl1c acts as a scaffold to orchestrate the activation and silencing of the spindle assembly checkpoint (SAC) signalling pathway, which delays cell cycle progression when chromosomes are not attached to the spindle. Knl1c and Ndc80c are bought together through concurrent and cooperative interactions with Mtw1c, whose interaction with inner kinetochore proteins is autoinhibited unless the kinetochore has active EC and SAC signalling pathways. The aim of this thesis was to determine the structural basis of KMN complex assembly and autoinhibition in the budding yeast S. cerevisiae. Using cryo-EM and biochemistry, I found that two uncharacterised -helices in C-terminal regions of the Mtw1 and Nnf1 subunits within Mtw1c contact distinct binding sites in Knl1c to mediate the interaction between Mtw1c and Knl1c. I also determined the structural basis by which a second -helix in the C-terminal extension of Mtw1 can bind to Ndc80c cooperatively with a well characterised -helix in the C-terminus of the Dsn1 subunit within Mtw1c. Using cryo-EM, biochemistry, biophysics, and protein structure predictions, I showed that an -helix in the N-terminal intrinsically disordered region of Dsn1 occludes the binding site in Mtw1c for the inner kinetochore protein CENP-CMif2. This thereby autoinhibits the interaction between Mtw1c and CENP-CMif2. Biophysics and biochemistry revealed that introducing mutations into this -helix that mimic phosphorylation at substrate sites for the EC pathway kinase Aurora B / Ipl1 reduced the affinity of the -helix for Mtw1c and relieved Mtw1c autoinhibition. These results therefore reveal the structural basis by which the S. cerevisiae KMN complex is assembled and recruited by CENP-CMif2 specifically to kinetochores with active signalling pathways.