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. 2014 Apr;42(7):4318-31.
doi: 10.1093/nar/gku090. Epub 2014 Feb 3.

DAXX co-folds with H3.3/H4 using high local stability conferred by the H3.3 variant recognition residues

Jamie E DeNizio  1 , Simon J Elsässer, Ben E Black
Affiliations

DAXX co-folds with H3.3/H4 using high local stability conferred by the H3.3 variant recognition residues

Jamie E DeNizio et al. Nucleic Acids Res. 2014 Apr.

Abstract

Histone chaperones are a diverse class of proteins that facilitate chromatin assembly. Their ability to stabilize highly abundant histone proteins in the cellular environment prevents non-specific interactions and promotes nucleosome formation, but the various mechanisms for doing so are not well understood. We now focus on the dynamic features of the DAXX histone chaperone that have been elusive from previous structural studies. Using hydrogen/deuterium exchange coupled to mass spectrometry (H/DX-MS), we elucidate the concerted binding-folding of DAXX with histone variants H3.3/H4 and H3.2/H4 and find that high local stability at the variant-specific recognition residues rationalizes its known selectivity for H3.3. We show that the DAXX histone binding domain is largely disordered in solution and that formation of the H3.3/H4/DAXX complex induces folding and dramatic global stabilization of both histone and chaperone. Thus, DAXX uses a novel strategy as a molecular chaperone that paradoxically couples its own folding to substrate recognition and binding. Further, we propose a model for the chromatin assembly reaction it mediates, including a stepwise folding pathway that helps explain the fidelity of DAXX in associating with the H3.3 variant, despite an extensive and nearly identical binding surface on its counterparts, H3.1 and H3.2.

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Figures

Figure 1.
Figure 1.
H3.3/H4 dimer is globally stabilized by DAXX upon heterotrimer formation. (A) Experimental scheme for comparing H/DX of (H3.3/H4)2 heterotetramer, H3.3/H4/DAXX heterotrimer complex, H3.2/H4/DAXX heterotrimer complex and DAXX monomer. The locations of the ribbon diagrams with all time points for each corresponding H/DX data set are listed. (B and C) H/DX data for the histones from (H3.3/H4)2 and H3.3/H4/DAXX. Each horizontal bar represents an individual peptide from (H3.3/H4)2 (B) or H3.3/H4/DAXX (C) and is color-coded for percent deuteration at each time point (101, 102, 103, 104 and 105 s) by individual stripes within each bar. Peptides are placed beneath schematics of the secondary structural elements of H3.3 or H4 from the crystal structures of (H3.3/H4)2 (B) [from the H3.3 nucleosome, PDB 3AV2; (27)] and H3.3/H4/DAXX (C) [PDB 4H9N; (15)], which are shown adjacent to the H/DX data from each respective complex. (D–I) Enlarged peptides from panel B (D–I, left) and panel C (D–I, right) are shown side-by-side.
Figure 2.
Figure 2.
H3.3 αN helix is stably folded in the H3.3/H4/DAXX heterotrimer complex. (A) The location of a H3.3 peptide (residues 51–59), spanning the αN helix, is shown in black on both the (H3.3/H4)2 (PDB 3AV2) and H3.3/H4/DAXX (PDB 4H9N) crystal structures. The heterotetramer structure is from the stable secondary structures existing within the nucleosome core particle (27,28), but our data indicate the αN helix of H3.3 is unfolded in heterotetramers in solution. (B) Comparison of H/DX for the peptide spanning residues 51–59 from both complexes over the time course. The maximum number of deuterons possible to measure by H/DX is shown by a black dotted line. (C) Side-by-side analysis of MS data for the indicated peptide from (H3.3/H4)2 (left) or H3.3/H4/DAXX (right). Dotted red and blue lines serve as guideposts to highlight the differences in m/z shifts between the two complexes. Black stars denote the centroid locations.
Figure 3.
Figure 3.
DAXX induces alterations to and prevents unfolding of the H3.3 α2-L2-α3 region in solution. (A) Deuterium exchange rate profile maps of peptides spanning H3.3 residues 103–126 in (H3.3/H4)2 (top) and H3.3/H4/DAXX (bottom). Schematics of the secondary structural features from the crystal structures of both protein complexes (Figure 1B and C, right) are shown, with the region of interest boxed and expanded below. The primary sequence and consensus exchange rate at each position are also shown. The first two residues of each peptide and prolines are boxed in dashed black lines because exchange of the first two backbone amide protons cannot be measured (30) and prolines lack amide protons. (B and C) MS data of two representative peptides, which are displayed as in Figure 2C. Both peptides from the (H3.3/H4)2 complex exhibit EX1 behavior (D and E). Comparison of H/DX for the indicated H3.3 peptides from each of the complexes. When data are biphasic, the reported number of deuterons is calculated from the average centroid value over the relative intensities of both the ‘open’ and ‘closed’ populations. The consensus exchange rates assigned in panel A are mapped onto either the (H3.3/H4)2 (PDB 3AV2) (F) or H3.3/H4/DAXX (PDB 4H9N) (G) crystal structures. Other portions of H3.3 are shown in gray. (F) H3.3 residues involved in the H3.3:H3.3′ four-helix bundle of (H3.3/H4)2 are shown. (G) H3.3 residues that establish contacts between H3.3 and DAXX in H3.3/H4/DAXX are highlighted.
Figure 4.
Figure 4.
DAXX completely rescues the fold of a mutant version of H3.3/H4. SEC coupled with MALS and H/DX-MS of wild-type and mutant complexes of H3.3/H4 both without (A–C) and in complex with (D–F) DAXX. (A) The size of (H3.3/H4)2 is 53 kDa and that of H3.37sub/H4 is 38 kDa. (D) The size of H3.3/H4/DAXX is 61 kDa and that of H3.37sub/H4/DAXX is 58 kDa. (B, C, E and F) The consensus levels of H/DX at 101 s for (H3.3/H4)2 (B), H3.37sub/H4 (C), H3.3/H4/DAXX (E) and H3.37sub/H4/DAXX (F) are mapped onto an H3.3/H4 dimer from either the H3.3 nucleosome (PDB 3AV2) (B and C) or H3.37sub/H4/DAXX complex [PDB 4H9S; (15)] (E and F) crystal structures. Regions of H3.3 and H4 that are destabilized in H3.37sub/H4 but are then stabilized when in complex with DAXX are highlighted as follows: the H3.3:H3.3′ tetramerization region is circled, and the H3.3 and H4 α1 helices are indicated by an arrowhead and arrow, respectively.
Figure 5.
Figure 5.
DAXX behaves essentially as an unfolded protein before binding H3.3/H4. H/DX data of peptides from the DAXX monomer (A) and in a heterotrimer complex with H3.3/H4 (B). Data are presented as in Figure 1B and C. Peptides spanning residues 226–235 (C) and 322–332 (D) are enlarged in the bottom right corner of panel B and their MS peptide spectra are shown, which are presented as in Figures 2 and 3. (E) The consensus exchange rate of each residue from the H3.3/H4/DAXX complex is mapped onto the crystal structure. Residues lacking any peptide coverage are colored gray.
Figure 6.
Figure 6.
Stability induced in DAXX by contacts with H3.3-specific residues. (A) Differences in primary sequence between H3.2 and H3.3 histone variants. H3.3 residue S31 differs from H3.2 but is not important for recognition by DAXX. In contrast, H3.3 residues A87, I89 and G90 differ from H3.2 and are important for specific recognition by DAXX (8). These residues are shown in black space fill in the H3.3/H4/DAXX crystal structure (PDB 4H9N) to highlight the surrounding environment. (B) Decreased protection from H/DX of DAXX in complex with H3.2- compared with the H3.3-containing heterotrimer complex at 105 s. The level of protection is determined by subtracting the percent deuteration of H3.2/H4/DAXX from that of H3.3/H4/DAXX for individual DAXX peptides, which are colored according to the legend. Gray represents no difference in H/DX between the two complexes, and white indicates the small number of positions lacking peptide coverage. Overlapping peptides at each position are assigned a consensus behavior, which is shown above the peptides. (C) The consensus difference at each residue is mapped onto DAXX from the H3.3/H4/DAXX crystal structure (PDB 4H9N). DAXX residues that line the area surrounding the H3.3-specificity residues are shown in space fill. (D) Comparison of H/DX for a DAXX peptide spanning L1. (E) Comparison of H/DX for a DAXX peptide spanning the α5 helix.
Figure 7.
Figure 7.
A stepwise co-folding model to explain how H3.3-specific assembly with DAXX is achieved. See text for details.
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