Molecular density-accelerated binding-site maturation underlies CENP-T-dependent kinetochore assembly

Formation of macromolecular cellular structures relies on recruitment of multiple proteins, requiring the precisely controlled pairwise binding interactions. At human kinetochores, our recent work found that the high molecular density environment enables strong bonding between the Ndc80 complex and its two binding sites at the CENP-T receptor. However, the mechanistic basis for this unusual density-dependent facilitation remains unknown. Here, using quantitative single-molecule approaches, we reveal two distinct mechanisms that drive preferential recruitment of the Ndc80 complex to higher-order structures of CENP-T, as opposed to CENP-T monomers. First, the Ndc80 binding sites within the disordered tail of the CENP-T mature over time, leading to a stronger grip on the Spc24/25 heads of the Ndc80 complexes. Second, the maturation of Ndc80 binding sites is accelerated when CENP-T molecules are clustered in close proximity. The rates of the clustering-induced maturation are remarkably different for two binding sites within CENP-T, correlating with different interfaces formed by the corresponding CENP-T sequences as they wrap around the Spc24/25 heads. The differential clustering-dependent regulation of these sites is preserved in dividing human cells, suggesting a distinct regulatory entry point to control kinetochore-microtubule interactions. The tunable acceleration of slowly maturing binding sites by a high molecular-density environment may represent a fundamental physicochemical mechanism to assist the assembly of mitotic kinetochores and other macromolecular structures.