The structural basis for kinetochore stabilization by Cnn1/CENP-T

Abstract

Chromosome segregation depends on a regulated connection between spindle microtubules and centromeric DNA. The kinetochore, a massive modular protein assembly, mediates this connection and also serves as a signaling hub that integrates and responds to changing cues during the cell cycle. Kinetochore functions evolve as the cell cycle progresses, culminating in the assurance of a persistent chromosome-microtubule connection during anaphase, when sister chromatids must transit into daughter cells uninterrupted. We previously determined the structure of the Ctf19 complex, a group of kinetochore proteins at the centromeric base of the kinetochore. We now present a high-resolution structure of a Ctf19 complex sub-assembly involved in centromere-microtubule contact: the Ctf3 complex bound to the Cnn1-Wip1 heterodimer. The resulting composite model of the Ctf19 complex and live-cell imaging experiments provide a mechanism for Cnn1-Wip1 recruitment to the kinetochore. The mechanism suggests feedback regulation of Ctf19 complex assembly and unanticipated similarities in kinetochore organization between yeast and vertebrates.

Assembly principles and stoichiometry of a complete human kinetochore module

“…In solid phase binding assays with immobilized MBP CENP-C F ( Figure 5A-B) we observed binding of CENP-TWSX in presence of CENP-HIKM and CENP-LN, reduced binding upon omission of CENP-LN, and poor binding upon omission of CENP-HIKM or of both CENP-HIKM and CENP-LN (shown schematically in Figure 5C). Thus, CENP-HIKM, and marginally also CENP-LN, provide a binding platform for CENP-TWSW, in agreement with previous studies in S. cerevisiae (Basilico et al, 2014;Hinshaw and Harrison, 2020;Pekgoz Altunkaya et al, 2016;Zhang et al, 2020). A highly conserved C-terminal helical hairpin of CENP-T (helices a4 and a5), which extends the HFD to complete a binding interface for the CENP-HIK complex (Hinshaw and Harrison, 2020;Zhang et al, 2020), was required for tight binding to the MBP CENP-C F -CCAN complex in vitro as well as for its kinetochore recruitment after electroporation ( Figure 5D-E and Figure S6F-G).…”

Crystal structure of the Cenp-HIKHead-TW sub-module of the inner kinetochore CCAN complex

“…We analysed the conformational variability of the Cenp-HIK Head -TW sub-module by comparing the conformations of Cenp-HIK-TW in the context of three states (i) the CCAN – Cenp-A Nuc complex ( 37 ), (ii) the CCAN dimer ( 37 ), and (iii) the recently reported Cenp-HIK-TW complex (Ctf19c-Cnn1-Wip1) ( 57 ). Superimposing all three structures onto the structurally invariant Cenp-HIK Body shows that Cenp-HIK Head -TW sub-module adopts a range of conformations facilitated by the flexible joint that connects Cenp-HIK Body and Cenp-HIK Head -TW (Figure 4C and Supplementary Figure S5 ).…”

“…While this manuscript was in preparation, the cryo-EM structure of the S. cerevisiae Ctf3c-Cnn1-Wip1 (Cenp-HIK-TW) complex was deposited (PDB: 6WUC) ( 57 ). In this structure, the Cenp-HIK–Cenp-TW interface, involving the HFE of Cenp-T is well defined, with conclusions similar to ours.…”

“…This was possible because a 3D cryo-EM class of the CCAN–Cenp-A complex (EMD-11626) revealed EM density for the Cenp-HIK Head -TW sub-module. The remaining subunits of CCAN are as reported previously ( 37 ), except that we used the recent cryo-EM model of S. cerevisiae Ctf3c–Cnn1–Wip1 (Cenp-HIK-TW) (PDB 6WUC) ( 57 ) to fit the ‘flexible ‘joint’ region that connects Cenp-HIK Body to CenpHIK Head : residues 139–142 of Cenp-H, residues 285–353 of Cenp-I, and residues 141–148 of Cenp-K as a rigid body fit in Coot ( 52 ), and additionally, the α-helix (residues 242–268) to Cenp-I Head . The fit of Cenp-HIK Head -TW into the CCAN – Cenp-A Nuc cryo-EM map (EMD-11626) was assessed using the Chimera-derived correlation coefficient between the calculated EM density of the fitted models and the cryo-EM map.…”

Multi-site phosphorylation of yeast Mif2/CENP-C promotes inner kinetochore assembly

“…The DDK step necessarily occurs before inner kinetochore stabilization and therefore in the absence of robust Ctf3 localization, which is required for maximal DDK localization (Hinshaw et al, 2017). Indeed, kinetochore-associated Ctf3-GFP intensity is at its nadir as cells approach S-phase (Hinshaw and Harrison, 2020). Thus, DDK-dependent Mif2-PEST phosphorylation produces more robust Ctf19c assembly, which in turn results in further Mif2-PEST phosphorylation.…”