The kinetochore is a proteinaceous complex that is essential for proper chromosome segregation. As a core member of the inner kinetochore, defects of each subunit in the CENP-H/I/K complex cause dysfunction of kinetochore that leads to chromosome mis-segregation and cell death. However, how the CENP-H/I/K complex assembles and promotes kinetochore function are poorly understood. We here determined the crystal structures of CENP-I N-terminus alone from Chaetomium thermophilum and its complex with CENP-H/K from Thielavia terrestris, and verified the identified interactions. The structures and biochemical analyses show that CENP-H and CENP-K form a heterodimer through both N- and C-terminal interactions. CENP-I integrates into the CENP-H/K complex by binding to the C-terminus of CENP-H, leading to formation of the ternary complex in which CENP-H is sandwiched between CENP-K and CENP-I. Our sequence comparisons and mutational analyses showed that this architecture of the CENP–H/I/K complex is conserved in human. Mutating the binding interfaces of CENP-H for either CENP-K or CENP-I significantly reduced their localizations at centromeres and induced massive chromosome alignment defects during mitosis, suggesting that the identified interactions are critical for CENP-H/I/K complex assembly at the centromere and kinetochore function. Altogether, our findings unveil the evolutionarily conserved assembly mechanism of the CENP-H/I/K complex that is critical for proper chromosome alignment.

Error-free mitosis depends on accurate chromosome attachment to spindle microtubules, which is monitored by the spindle assembly checkpoint (SAC) signaling. As an upstream factor of SAC, the precise and dynamic kinetochore localization of Mps1 kinase is critical for initiating and silencing SAC signaling. However, the underlying molecular mechanism remains elusive. Here, we demonstrated that the multisite interactions between Mps1 and Ndc80 complex (Ndc80C) govern Mps1 kinetochore targeting. Importantly, we identified direct interaction between Mps1 tetratricopeptide repeat domain and Ndc80C. We further identified that Mps1 C-terminal fragment, which contains the protein kinase domain and C-tail, enhances Mps1 kinetochore localization. Mechanistically, Mps1 C-terminal fragment mediates its dimerization. Perturbation of C-tail attenuates the kinetochore targeting and activity of Mps1, leading to aberrant mitosis due to compromised SAC function. Taken together, our study highlights the importance of Mps1 dimerization and multisite interactions with Ndc80C in enabling responsive SAC signaling.