(a) Binding of in vitro translated ³⁵S-labeled HA-HIRA(421–729) and the indicated B-domain deletion mutants to ³⁵S-labeled Myc-ASF1a.
(b) Binding of in vitro translated ³⁵S-labeled GFP-HIRA(421–729) and the indicated C-terminal deletion mutants to ³⁵S-labeled Myc-ASF1a.
(c) Binding of in vitro translated ³⁵S-labeled HA-HIRA(421–729) to ³⁵S-labeled Myc-ASF1a in the presence of 0, 1, 10 and 50µg of the wild-type HIRA peptide, RTADGRRRITPLCIA, or 50µg of the scrambled peptide, GRAARITPRDTLRCI. The height of the square and triangle indicates relative peptide concentrations.

(a) Close-up view of the ASF1a/HIRA interface. ASF1a is shown as a gold ribbon in combination with a semi-transparent surface representation and HIRA as a green ribbon. Residues from both molecules that mediate hydrogen-bonds (including salt-bridges) and hydrophobic interactions are highlighted as sticks with CPK coloring. Hydrogen bonds are shown with dashed lines (yellow for intra-molecular and red for inter-molecular).
(b) Surface electrostatic representation of ASF1a and HIRA moieties in the complex. For clarity, the HIRA fragment in the complex is shown only as sticks with CPK coloring (right), with the HIRA surface shown in an open-book format (left). Residues involved in salt-bridge interaction are indicated on the corresponding surface. This figure was made with GRASP41.

(a) Surface representation of the ASF1a/HIRA complex, with residues 1–30 of ASF1a highlighted in blue. The color-code highlights the sequence variation between ASF1a and ASF1b as indicated in the color bar. The HIRA fragment bound to ASF1a is also shown in green surface representation for reference.
(b) Hydrophobic residues (in yellow) are mapped onto a ribbon representation of ASF1a, with the cluster of hydrophobic residues at the histone H3 binding site31 shown as a space-filling model. The HIRA fragment bound to ASF1a is shown as a green surface representation for reference and the published NMR data is used to model a histone H3 C-terminal helix (in red) onto the ASF1a surface. The relative orientations between the left and right panels are indicated.
(c) A schematic model for ASF1a and ASF1b association with HIRA, CAF-1 p60 and histone H3. See text for details.

(a) Left- Binding of in vitro translated ³⁵S-labeled HA-HIRA(421–729) or the indicated mutants to ³⁵S-labeled Myc-ASF1a. Right- Binding of in vitro translated ³⁵S-labeled HA-HIRA(421–729) to ⁵S-labeled Myc-ASF1a or the indicated mutants.
(b) Representative data from isothermal titration calorimetry titrations of HIRA B-domain peptide (residues 453–467) titrated into ASF1aN (residues 1–154), ASF1bN (residues 1–154), and ASF1aFL (full length, residues 1–204). The area under each injection spike (upper section) is integrated and fitted by using nonlinear least-squares regression analysis (lower section). The derived enthalpies (ΔH), dissociation constant (K_d) and stoichiometry (n) are indicated for each of the titrations. This figure was prepared using Origin 5.0.
(c) Schematic of the protein chimaeras used in (d).
(d) Left - Binding of in vitro translated ³⁵S-labeled HA-HIRA(421–729) to the indicated ³⁵S-labeled ASF1 proteins and their C-termini (155–202) swapped chimaeras. Right - Binding of in vitro translated ³⁵S-labeled HA-HIRA(421–729) to the indicated ³⁵S-labeled ASF1 proteins and their N-termini (1–30) swapped chimaeras.

(a) Binding of the HIRA B-domain and selected mutants as GST fusion proteins to purified ASF1aN inputs.
(b) Binding of affinity purified human CAF-1 p60 and yeast Cac2 as GST fusion proteins to purified ASF1aN proteins.
(c) Binding of affinity purified CAF-1 p60 GST fusion proteins and their selected mutants to purified ASF1aN proteins.
(d) Physical interactions between ectopically expressed CAF-1 p60 and ASF1a proteins in human U2OS cells as tested by immunoprecipitation-western blot analysis. Left – Interaction between HA-tagged CAF-1 p60 wild-type or truncation mutant p60(1–481) and Myc-tagged wild-type ASF1a. Right – Interaction between HA-tagged CAF-1 p60 wild-type or mutant p60(RR482AA) or p60(RR498AA) with Myc-tagged wild-type ASF1a.
(e) Binding of bacterially expressed and affinity purified HIRA (427–472) and CAF-1 p60 (376–559) GST fusion proteins to purified ASF1aN, ASF1bN, ASF1a full-length or ASF1aN(D37A) mutant proteins.
(f) Disruption of CAF-1 p60 (469–559)/ASF1aN and HIRA (427–472)/ASF1aN complexes by a HIRA (453–467) peptide. In (b), (c), (e) and (f), only the upper (intact) band of each individual GST fusion protein was used for evaluation of binding to ASF1a because of partial proteolysis of the fusion proteins during expression and purification.

(a) Ribbon representation of the ASF1a/HIRA complex. ASF1a is in yellow, with the three helices in red and the bound HIRA fragment in green.
(b) Section of the composite-omit electron density map (contoured at 1.0 σ) of the ASF1a/HIRA complex crystal, showing a region of the HIRA fragment.
(c) Cα superposition of the two ASF1a/HIRA complexes (ASF1a in yellow and HIRA green) within the asymmetric unit, with the nascent structures of yeast Asf1p (red) and human ASF1a (blue). The black box defines the close-up region in (d).
(d) A close-up view of the superposition of the structures in (c) around the ASF1a VGP (residues 62–64) triplet. Only one complex of the two ASF1aN/HIRA complexes in the asymmetric unit cell, highlighted with a ball-and-stick model, is displayed for clarity. The color-coding is as described in (c) with CPK coloring and hydrogen bonds shown. Fig. 3, 4a and 7 were created with Pymol (DeLano W.L.; www.pymol.org).

(a) The extended B-domain of HIRA homologues are aligned with strictly conserved (identical) residues shaded in red and conserved residues shaded in yellow, with the minimal HIRA peptide required for ASF1a binding underlined. The blue box indicates the core B-domain, defined previously by sequence analysis and functional studies18,32,33. The residue numbering and the secondary structure for human HIRA are indicated above the sequence alignment. Ordered regions of HIRA in the crystal structure are indicated by the solid black line above the alignment and disordered regions are indicated with a dashed line. Residues of HIRA that make contacts to ASF1a are indicated with blue symbols (arrowhead - salt bridge interaction; oval - hydrophobic interaction; star - structural and/or non-salt bridge hydrogen bonding). Abbreviations for various organisms are Bt, B. Taurus; Ca, C. albicans; Ce, C. elegans; Dm, D. melanogaster; Gg, G. gallus; Hs, H. sapiens; Mm, M. musculus; Pt, P. troglodytes; Sc, S. cerevisiae; and Sp, S. pombe;
(b) Sequence alignment of the B-domain-like motifs of human CAF-1 p60 and its homologues from other species. For the proteins that have two motifs, both of them are given separately with (1) and (2) designations. The human HIRA B-domain is shown on the top for comparison. Residues of HIRA that make contacts to ASF1a are numbered on the top and indicated below the alignment using the same symbols described in (A). Starting and ending residue numbers are marked on the sequence, with the total number of residues in the full-length proteins indicated at the end of the sequences. Abbreviations for various organisms are as in (A).