Structure of the MIS12 Complex and Molecular Basis of Its Interaction with CENP-C at Human Kinetochores

Abstract

SummaryKinetochores, multisubunit protein assemblies, connect chromosomes to spindle microtubules to promote chromosome segregation. The 10-subunit KMN assembly (comprising KNL1, MIS12, and NDC80 complexes, designated KNL1C, MIS12C, and NDC80C) binds microtubules and regulates mitotic checkpoint function through NDC80C and KNL1C, respectively. MIS12C, on the other hand, connects the KMN to the chromosome-proximal domain of the kinetochore through a direct interaction with CENP-C. The structural basis for this crucial bridging function of MIS12C is unknown. Here, we report crystal structures of human MIS12C associated with a fragment of CENP-C and unveil the role of Aurora B kinase in the regulation of this interaction. The structure of MIS12:CENP-C complements previously determined high-resolution structures of functional regions of NDC80C and KNL1C and allows us to build a near-complete structural model of the KMN assembly. Our work illuminates the structural organization of essential chromosome segregation machinery that is conserved in most eukaryotes.

Molecular Structures of Yeast Kinetochore Subcomplexes and Their Roles in Chromosome Segregation

“…Both these interactions are sensitive to phosphorylation of residues in a Dsn1 aminoterminal extension that projects from head 2 (Akiyoshi et al 2013). When not phosphorylated, this segment of Dsn1 binds head 1 and blocks the sites for Mif2 and Ame1 Petrovic et al 2016). Phosphorylation at two positions (serines 240 and 250 in Saccharomyces cerevisiae) by Ipl1…”

“…Thus, Cnn1:Wip1, presumably with associated Ndc80 complexes, may enhance kinetochore strength or connectivity but probably does not modify its fundamental molecular organization. The two adaptors bind Spc24:Spc25 through related motifs-at the carboxy-terminal end of the MIND subunit Dsn1 and near the aminoterminal end of Cnn1 (Bock et al 2012;Malvezzi et al 2013;Dimitrova et al 2016;Petrovic et al 2016). Mps1 phosphorylation of a serine residue in the binding segment of Cnn1 blocks the interaction (Thapa et al 2015); serine residues in the related Dsn1 segment are potential sites of phosphoregulation, but a role for their modification, if any, has yet to be identified .…”

“…MIND is a 210-Å-long, heterotetrameric rod, with two globular, four-helix bundles ("heads") at one end Petrovic et al 2016). Mtw1 MIS12 and Nnf1…”

How Kinetochore Architecture Shapes the Mechanisms of Its Function

“…B (left to right = microtubule plus-end to centromere) The conserved, dual pathways (solid arrows – direct interaction, dashed arrow – indirect interaction) that assemble the KMN network, which forms the interface of the kinetochore with the microtubule plus-end. C Reconstruction of the protein architecture of the budding yeast kinetochore using fluorescence microscopy measurements and protein structures [7, 8, 14, 15, 32, 33, 48–50, 73]. Centromere-associated proteins are represented by white, oblong shapes.…”

Anaphase A: Melting Microtubules Move Chromosomes toward Spindle Poles

“…Coupling performance improved with the incorporation of additional microtubule-binding kinetochore elements [153,154], with the use of native kinetochore particles isolated from yeast [43], and with the use of flexible tethers for linking sub-complexes to beads [155]. Further improvements seem likely, especially as continued advancements in kinetochore biochemistry enable reconstitutions of ever more complete and stable kinetochore assemblies [156][157][158]. However, the performance achieved in laser trap tip-coupling assays already provides a reasonably good match to physiological conditions.…”