Cryo‐EM structure of the CENP‐A nucleosome in complex with phosphorylated CENP‐C

Abstract

The CENP‐A nucleosome is a key structure for kinetochore assembly. Once the CENP‐A nucleosome is established in the centromere, additional proteins recognize the CENP‐A nucleosome to form a kinetochore. CENP‐C and CENP‐N are CENP‐A binding proteins. We previously demonstrated that vertebrate CENP‐C binding to the CENP‐A nucleosome is regulated by CDK1‐mediated CENP‐C phosphorylation. However, it is still unknown how the phosphorylation of CENP‐C regulates its binding to CENP‐A. It is also not completely understood how and whether CENP‐C and CENP‐N act together on the CENP‐A nucleosome. Here, using cryo‐electron microscopy (cryo‐EM) in combination with biochemical approaches, we reveal a stable CENP‐A nucleosome‐binding mode of CENP‐C through unique regions. The chicken CENP‐C structure bound to the CENP‐A nucleosome is stabilized by an intramolecular link through the phosphorylated CENP‐C residue. The stable CENP‐A‐CENP‐C complex excludes CENP‐N from the CENP‐A nucleosome. These findings provide mechanistic insights into the dynamic kinetochore assembly regulated by CDK1‐mediated CENP‐C phosphorylation.

Assembly principles and stoichiometry of a complete human kinetochore module

“…CENP-N interacts directly with CENP-A NCPs by recognizing the CENP-A L1 loop ( 14 , 40 , 43 , 44 ). CENP-C, on the other hand, interacts with the acidic patch of H2A-H2B and with the CENP-A C-terminal tail ( 8 , 9 , 11 , 15 , 36 , 40 , 44 , 45 ). These features may allow CENP-C and CENP-N to bind concomitantly to their cognate binding sites on the same CENP-A nucleosome, although the interaction may be further regulated by phosphorylation ( 44 , 45 ).…”

“…CENP-C, on the other hand, interacts with the acidic patch of H2A-H2B and with the CENP-A C-terminal tail ( 8 , 9 , 11 , 15 , 36 , 40 , 44 , 45 ). These features may allow CENP-C and CENP-N to bind concomitantly to their cognate binding sites on the same CENP-A nucleosome, although the interaction may be further regulated by phosphorylation ( 44 , 45 ). Furthermore, an alternative binding model where CENP-N occupies a noncanonical position has also been recently proposed ( 8 ).…”

“…Our observation that CENP-C F binds selectively to CENP-A nucleosomes over H3 nucleosomes even at micromolecular concentrations in vitro was unexpected, as previous studies had reported that at least the CENP-C motif, when isolated from the rest of CENP-C, binds relatively nonselectively to CENP-A and H3 NCPs ( 11 , 36 ), although another study failed to detect an interaction of the CENP-C motif with H3.3 nucleosomes ( 65 ). Phosphorylation of CENP-C near the CENP-C motif has been recently shown to facilitate the interaction with CENP-A NCPs in vitro and CENP-C kinetochore localization ( 17 , 45 ). The interaction of CENP-C with two nucleosomes, implied by the presence of CENP-A NCP binding motifs in the central region and the CENP-C motifs, had been postulated ( 2 , 11 ) but remained undemonstrated.…”

CENP-N promotes the compaction of centromeric chromatin

“…high-density sucrose fractions, fig. S11D, G, fractions [15][16][17][18][19][20] and that this compaction state corresponds to the presence of the CENP-C protein. Altogether, our in vivo studies validate the in vitro results to demonstrate that CENP-N plays an important role in compaction of CENP-A nucleosomes through its α-6 helix.…”

“…The key function of CENP-A nucleosomes appears to be the recruitment of centromere-specific proteins, most notably CENP-N and CENP-C, both of which recognize unique features of nucleosomal CENP-A, and upon which the CCAN complex and ultimately the kinetochore assemble (reviewed in (11)). Both CENP-N and CENP-C affect CENP-A nucleosome dynamics and structure in vitro at the mono-nucleosome level (12)(13)(14)(15)(16)(17), but their effect on chromatin higher order structure has not been investigated at the molecular level (18).…”