Structure of a CENP-A-histone H4 heterodimer in complex with chaperone HJURP

Abstract

In higher eukaryotes, the centromere is epigenetically specified by the histone H3 variant Centromere Protein-A (CENP-A). Deposition of CENP-A to the centromere requires histone chaperone HJURP (Holliday junction recognition protein). The crystal structure of an HJURP-CENP-A-histone H4 complex shows that HJURP binds a CENP-A-H4 heterodimer. The C-terminal β-sheet domain of HJURP caps the DNA-binding region of the histone heterodimer, preventing it from spontaneous association with DNA. Our analysis also revealed a novel site in CENP-A that distinguishes it from histone H3 in its ability to bind HJURP. These findings provide key information for specific recognition of CENP-A and mechanistic insights into the process of centromeric chromatin assembly.

Figure 1.

Overall structure. (A) A ribbon diagram of the complex structure. CENP-A is shown in green, histone H4 is shown in cyan, and HJURP is shown in yellow. Secondary structure elements in CENP-A, H4, and HJURP are labeled in black, white, and red letters, respectively. The shaded area indicates the CATD of CENP-A. (B) Overview of interactions between HJURP and the CENP-A–H4 heterodimer. The structure is viewed from the same direction as in A, and the heterodimer is shown in a surface representation with electrostatic potential distribution; white, blue, and red regions indicate neutral areas, positively charged areas, and negatively charged areas, respectively. The side chains of HJURP are shown in a stick model (carbon, yellow; nitrogen, blue; oxygen, red; sulfur, gold) superimposed on the ribbon representation of the main chain. An inset enclosed in the red dashed circle on the right indicates an orthogonal view of an interaction region (enclosed in the left circle) involving amino acids in the L1 and β-sheet domain. CENP-A residues involved in the interactions in this region are also shown in a stick model. (Green) Carbon. White and black letters label selected CENP-A and HJURP residues, respectively.

Figure 2.

Structural comparison of heterodimeric and tetrameric CENP-A–H4 complexes. The CENP-A–H4 complex from the cocrystal structure with HJURP is colored green (CENP-A) and cyan (histone H4), and the same complex from the CENP-A–H4 tetramer is shown in magenta (CENP-A) and pale cyan (histone H4). The structures are viewed from a direction opposite to that in Figure 1A. For viewing clarity, the HJURP molecule is not shown. Four regions of main differences are enclosed in orange dashed-line circles, and the regions are numbered according to the order in which they were referenced in the text. An inset encircled with a solid orange line represents an enlarged view of region 2, and relevant amino acids are shown in a stick model.

Figure 3.

HJURP binding prevents the formation of a CENP-A–histone H4 tetramer. (A) The structure of the HJURP–CENP-A–H4 complex is superimposed with the structure of the CENP-A–H4 tetramer (PDB ID: 3NQJ). (Left) The heterodimer from the tetramer structure superimposed with the HJURP complex is shown in a ribbon representation (CENP-A, magenta; H4, pale cyan), and the ribbon representation of the other heterodimer (CENP-A, light pink; H4, pale cyan) is overlaid with a surface representation. The HJURP complex is colored the same as in Figure 1A. (B) Histone chaperone Asf1 employs a different mechanism to block histone H3–H4 tetramer formation. The structure of the Asf1 complex (PDB ID: 2HUE) is shown in a ribbon diagram. (Yellow) Asf1; (magenta) H3; (pale cyan) H4. The H3–H4 dimer is aligned with the CENP-A–H4 heterodimer from the HJURP complex, the latter is positioned the same as in A but with HJURP removed.

Figure 4.

DNA-binding effect of HJURP. (A) Position of the nucleosomal DNA with one of the H3–H4 heterodimers (PDB code: 1KX5) aligned with the HJURP complex. The shaded area indicates the β-sheet and L1 regions that obstruct the potential path of nucleosomal DNA in a CENP-A-containing nucleosome. (B) HJURP prevents spontaneous DNA binding. (Lane 1) Free 208-bp α-satellite DNA probe. (Lane 2) Spontaneous binding of CENP-A–H4 to DNA. (Lanes 3–5) Increasing amount of GST-HJURP reduces binding to DNA. (Lanes 6, 10) GST-HJURP or GST alone do not bind DNA. (Lanes 7–9) GST does not affect DNA binding. An asterisk indicates the position of a protein–DNA complex formed with the addition of HJURP and the CENP-A–H4 complex. Please note that the intensity of this band is stronger at a 1:1 than at higher HJURP-to-CENP-A–H4 molar ratios.

Figure 5.

Determinants of CENP-A specificity. (A) His 104 and Gln 89, shown in the CATD of CENP-A (α2 and loop-1), make CENP-A-specific contacts with HJURP. Histone H3 (magenta) of a H3–H4 heterodimer (PBD ID: 1KX5) is superimposed with CENP-A. CENP-A residues making specific contacts, H3 residues in corresponding positions, and HJURP residues involved in the interactions are shown in a stick model. Selected residues of CENP-A, H3, and HJURP are labeled with blue, magenta, and black letters, respectively. An inset encircled with a red dashed line shows the interaction site of Ser 68 of CENP-A located outside of the CATD and surrounding HJURP residues. The atomic radii of side chain atoms of the Ser 68 counterpart in H3, Gln 68, and HJURP residues are shown in a dot model to indicate unfavorable packing of Gln 68. (B) GST pull-down experiments showing the effect of HJURP binding to CENP-A with Ser 68 mutations. (C) GST pull-downs with the H3–H4, H3(Q68S)–H4, H3^CATD–H4, and CENP-A–H4 complexes. To lower the background level of H3–H4 binding, 0.05% NP-40 in the washing buffer was used, compared with 0.025% NP-40 in B.