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Structural mechanisms of centromeric nucleosome recognition by the kinetochore protein CENP-N

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Science  19 Jan 2018:
Vol. 359, Issue 6373, pp. 339-343
DOI: 10.1126/science.aar2781
  • Fig. 1 Structure of the human CENP-N/CENP-A nucleosome complex.

    (A) Cryo-EM density map of the hCENP-N1–286/CENP-A nucleosome complex viewed down the axis of the DNA supercoil. (B) Schematic of the functional domains of CENP-N known to bind the CENP-A nucleosome (gray) and CENP-L (black) (top panel). The CENP-N construct used for the present structural analysis (hCENP-N1–286) and the regions of the sequence whose structure we report here [N-terminal domain: residues 1 to 81, and central domain: residues 101 to 185; hCENP-N(1–185)] are shown in the middle and bottom panels, respectively. (C) Cryo-EM density map of the hCENP-N1–286/CENP-A nucleosome complex as viewed from the side, at an orientation 90° to the view shown in (A). This view also depicts the extra density connected to the N-terminal domain that we assign to MBP, shown with lighter shading. (D) Representative regions of the cryo-EM density map to illustrate map quality (from left to right) for canonical histones H2A, H2B, and H4, centromere-specific H3 variant CENP-A, nucleosomal DNA, and CENP-N.

  • Fig. 2 Interaction of CENP-N with nucleosomal DNA.

    (A) Cut-away view of the hCENP-N1–286/CENP-A nucleosome model to highlight interfaces involved in complex formation (see also fig. S11, A and B). For the CENP-N/DNA interface (labeled “1a” and “1b”), nucleosomal DNA is shown as a red ribbon, whereas positively charged residues of CENP-N that are proposed to interact with it are shown as blue spheres. For the CENP-N/CENP-A interface (labeled “2”), CENP-A residues (R80, G81, and V82) are marked by the short yellow ribbon, whereas interacting CENP-N residues (E3, T4, and E7) are shown as yellow spheres. (B) View of the CENP-N/DNA interface at different magnifications to highlight details of interactions between the nucleosomal DNA and positively charged residues of CENP-N. (C) Gel mobility shift experiment to examine the effects of CENP-N mutations (indicated atop the gel) on binding to the CENP-A nucleosome. Impaired binding is reflected by increased intensity of the free nucleosome (Nuc) band, concomitant with the disappearance of defined 1:1 and 2:1 bands. “N” indicates the migration position of the free CENP-A nucleosome; “1” and “2” denote the migration positions of CENP-A nucleosomes bound with either one or two molecules of CENP-N, respectively. WT, wild type. (D) Similar analysis to that in (C), carried out with a set of CENP-N mutations involving residues distal from the binding interface. (E) Images of interphase nuclei in Xenopus egg extracts with exogenous MBP-xCENP-N and xCENP-L proteins containing the indicated mutations (with analogous human mutations in parentheses), stained with an antibody for MBP (green) and Hoechst (blue). (F) Centromeric MBP fluorescence intensity normalized as a percentage of that observed for wild-type MBP-xCENP-N. Error bars represent SEM (n > 200 centromeres). A.U., arbitrary units.

  • Fig. 3 Interaction between the L1 loop of CENP-A and helix 1 of CENP-N.

    (A and B) Overall (A) and close-up (B) view of the hCENP-N1–286/CENP-A interface formed between R80, G81, and V82 on the L1 loop of CENP-A and E3, T4, and E7 on helix 1 of CENP-N. (C) Gel mobility shift experiment to examine the effects of CENP-N mutations (indicated atop the gel) on binding to the CENP-A nucleosome. (D) Images of interphase nuclei in Xenopus egg extracts with exogenous MBP-xCENP-L and xCENP-L proteins containing the indicated mutations of xCENP-N residues E21 and E25 (corresponding to residues E3 and E7 in hCENP-N), stained with an antibody for MBP (green) and Hoechst (blue). (E) Centromeric MBP fluorescence intensity normalized as a percentage of that observed for wild-type MBP-xCENP-N. Error bars represent SEM (n > 200 centromeres). (F) Alignment of human and Xenopus laevis sequences corresponding to the L1 loop of CENP-A and helix 1 of CENP-N. Closely interacting segments of the L1 loop of CENP-A and helix 1 of CENP-N are highlighted by the shaded areas. The asterisks indicate conserved glutamic acid residues (black asterisks) and variability in the hydrophobic residue corresponding to position T4 (red asterisk) of human CENP-N. (G) Images of interphase nuclei in Xenopus egg extracts with exogenous MBP-xCENP-N and xCENP-L proteins containing the indicated mutations of xCENP-N, as in (D). (H) Centromeric MBP fluorescence intensity, determined as in (E). (I) Gel mobility shift experiment to examine the effects of correlated amino acid substitutions between the L1 loop of CENP-A and helix 1 of CENP-N.

  • Fig. 4 Structural determinants of kinetochore assembly on the CENP-A nucleosome.

    (A) Sequence alignment between human H3.1 and CENP-A to highlight distinct CENP-A motifs involved in deposition and recognition of CENP-A at centromeric chromatin (see also fig. S11, C and D). Single-letter abbreviations for the amino acid residues are as follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr. (B) Two different views of the CENP-A nucleosome bound to hCENP-N and a modeled CENP-C motif peptide (5) to highlight potential dual binding of full-length CENP-C and CENP-N proteins on the CENP-A nucleosome. The second CENP-N (shown with lighter shading) is modeled on the basis of the cryo-EM density map obtained in the presence of excess hCENP-N1–286 (fig. S4), whereas the CENP-C motif peptides (human numbering shown for clarity) on each face of the nucleosome are positioned according to the crystal structure of the nucleosome in complex with the rat CENP-C motif (5). (C) Schematic view to highlight recognition and possible enrichment of CENP-A nucleosomes by the CCAN proteins CENP-C, CENP-N, and CENP-L. Other kinetochore proteins and the dimerization of CENP-C have been omitted for clarity.

Supplementary Materials

  • Structural mechanisms of centromeric nucleosome recognition by the kinetochore protein CENP-N

    Sagar Chittori, Jingjun Hong, Hayden Saunders, Hanqiao Feng, Rodolfo Ghirlando, Alexander E. Kelly, Yawen Bai, Sriram Subramaniam

    Materials/Methods, Supplementary Text, Tables, Figures, and/or References

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    • Materials and Methods
    • Figs. S1 to S15
    • Table S1
    • References

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