A conserved mechanism for centromeric nucleosome recognition by centromere protein CENP-C

Abstract

Chromosome segregation during mitosis requires assembly of the kinetochore complex at the centromere. Kinetochore assembly depends on specific recognition of the histone variant CENP-A in the centromeric nucleosome by centromere protein C (CENP-C). We have defined the determinants of this recognition mechanism and discovered that CENP-C binds a hydrophobic region in the CENP-A tail and docks onto the acidic patch of histone H2A and H2B. We further found that the more broadly conserved CENP-C motif uses the same mechanism for CENP-A nucleosome recognition. Our findings reveal a conserved mechanism for protein recruitment to centromeres and a histone recognition mode whereby a disordered peptide binds the histone tail through hydrophobic interactions facilitated by nucleosome docking.

Figure 1

Binding sites between the CENP-C central region on the chimeric nucleosome mapped by methyl-based NMR. (A) Histone methyl chemical shift perturbation (CSP) upon CENP-C_426-537 binding. (B) Effects on methyl groups by paramagnetic spin labels in CENP-C_484-537. (C) Nucleosome structure showing representative side chains (black balls) whose methyl groups display large changes in (A) or (B), and approximate locations of CENP-C residues (arrows). E133 in H3 is used to represent L133 in H3_1-132-LEEGLG.

Figure 2

Residues important for binding of the central region of CENP-C and the chimeric nucleosome. (A) Effects of mutations in CENP-C_426-537 on its binding to the H3_1-132-LEEGLG nucleosome. No detectable binding (*). (B) Effect of CENP-C_1-537 mutations on centromere targeting in human cells (fig. S8B). Error represents SEM n=4, Students t probability p≤0.02 for all mutants. (C) Effect of H2A acidic patch mutations on localization of CENP-C to the CENP-A nucleosome arrays. Error represents SEM, n=4, Students t probability p≤0.05 for both mutants.

Figure 3

Broadly conserved CENP-C motifs bind to the H3 nucleosome chimera containing CenH3 C-terminal tails. (A) Nucleosome methyl CSP upon binding of the CENP-C motif. (B) The structure of the rat CENP-C motif in complex with the H3_1-132-IEGGLG nucleosome. (C) Enlarged region showing hydrophobic and electrostatic interactions.

Figure 4

Hydrophobicity of the CenH3 tail is the primary determinant for recognition of CenH3 by CENP-C. Isothermal titration calorimetric curves for binding of the human CENP-C central region (CENP-C_444-537) and the CENP-C motif (CENP-C_727-767) to nucleosomes containing H3_1-132-LEEGLG (human) and H3_1-132-QFI (budding yeast), respectively.