Faithful chromosome segregation requires robust, load-bearing attachments of chromosomes to the mitotic spindle, a function accomplished by large macromolecular complexes termed kinetochores. In most eukaryotes, the constitutive centromere-associated network (CCAN) complex of the inner kinetochore recruits to centromeres the ten-subunit outer kinetochore KMN network that comprises the KNL1C, MIS12C and NDC80C complexes. The KMN network directly attaches CCAN to microtubules through MIS12C and NDC80C. Here, we determined a high-resolution cryo-EM structure of the human KMN network. This showed an intricate and extensive assembly of KMN subunits, with the central MIS12C forming rigid interfaces with NDC80C and KNL1C, augmented by multiple peptidic inter-subunit connections. We also observed that unphosphorylated MIS12C exists in an auto-inhibited state that suppresses its capacity to interact with CCAN. Ser100 and Ser109 of the N-terminal segment of the MIS12C subunit Dsn1, two key targets of Aurora B kinase, directly stabilize this auto-inhibition. Our study indicates how selectively relieving this auto-inhibition through Ser100 and Ser109 phosphorylation might restrict outer kinetochore assembly to functional centromeres during cell division.

忠实的染色体分离需要染色体与有丝分裂纺锤体牢固、承载地连接，这一功能是由称为着丝粒的大分子复合物完成的。在大多数真核生物中，内着丝粒的组成性着丝粒相关网络 (CCAN) 复合体将由 KNL1C、MIS12C 和 NDC80C 复合体组成的十亚基外着丝粒 KMN 网络招募到着丝粒。KMN网络通过MIS12C和NDC80C直接将CCAN附着到微管上。在这里，我们确定了人类 KMN 网络的高分辨率冷冻电镜结构。这表明 KMN 亚基错综复杂且广泛组装，中央 MIS12C 与 NDC80C 和 KNL1C 形成刚性界面，并通过多个肽亚基间连接增强。我们还观察到未磷酸化的 MIS12C 处于自动抑制状态，抑制其与 CCAN 相互作用的能力。MIS12C 亚基 Dsn1 N 端片段的 Ser100 和 Ser109 是 Aurora B 激酶的两个关键靶标，可直接稳定这种自动抑制。我们的研究表明，通过 Ser100 和 Ser109 磷酸化选择性地缓解这种自身抑制可能会在细胞分裂过程中限制外着丝粒组装到功能性着丝粒上。