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. 2007 Mar 13;104(11):4285-90.
doi: 10.1073/pnas.0603762104. Epub 2007 Mar 6.

Relationship between the structure of SET/TAF-Ibeta/INHAT and its histone chaperone activity

Shinsuke Muto  1 , Miki Senda, Yusuke Akai, Lui Sato, Toru Suzuki, Ryozo Nagai, Toshiya Senda, Masami Horikoshi
Affiliations

Relationship between the structure of SET/TAF-Ibeta/INHAT and its histone chaperone activity

Shinsuke Muto et al. Proc Natl Acad Sci U S A. .

Abstract

Histone chaperones assemble and disassemble nucleosomes in an ATP-independent manner and thus regulate the most fundamental step in the alteration of chromatin structure. The molecular mechanisms underlying histone chaperone activity remain unclear. To gain insights into these mechanisms, we solved the crystal structure of the functional domain of SET/TAF-Ibeta/INHAT at a resolution of 2.3 A. We found that SET/TAF-Ibeta/INHAT formed a dimer that assumed a "headphone"-like structure. Each subunit of the SET/TAF-Ibeta/INHAT dimer consisted of an N terminus, a backbone helix, and an "earmuff" domain. It resembles the structure of the related protein NAP-1. Comparison of the crystal structures of SET/TAF-Ibeta/INHAT and NAP-1 revealed that the two proteins were folded similarly except for an inserted helix. However, their backbone helices were shaped differently, and the relative dispositions of the backbone helix and the earmuff domain between the two proteins differed by approximately 40 degrees . Our biochemical analyses of mutants revealed that the region of SET/TAF-Ibeta/INHAT that is engaged in histone chaperone activity is the bottom surface of the earmuff domain, because this surface bound both core histones and double-stranded DNA. This overlap or closeness of the activity surface and the binding surfaces suggests that the specific association among SET/TAF-Ibeta/INHAT, core histones, and double-stranded DNA is requisite for histone chaperone activity. These findings provide insights into the possible mechanisms by which histone chaperones assemble and disassemble nucleosome structures.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Functional activities of SET/TAF-Iβ/INHATΔC. (A) Coomassie brilliant blue staining of purified SET/TAF-Iβ/INHAT WT (lane 1) and ΔC (lane 2) proteins. (B) Complex formation of SET/TAF-Iβ/INHAT with core histones. After incubating histones H2A–H2B, H3, and H4, or all four core histones with Ni-NTA agarose beads, which captured SET/TAF-Iβ/INHAT WT (lanes 1–3), ΔC (lanes 4–6), and no protein (lanes 7–9), the bead-bound fraction was resolved by SDS/PAGE and stained with Coomassie brilliant blue. (C) Interaction of SET/TAF-Iβ/INHAT WT (lane 1) and ΔC (lane 2) proteins with histone–agarose. (D) Histone binding specificity of SET/TAF-Iβ/INHAT WT and ΔC by competition assay. Shown is eluted SET/TAF-Iβ/INHAT in supernatant after addition of 100 pmol (lanes 1 and 4), 350 pmol (lanes 2 and 5), and 1,000 pmol (lanes 3 and 6) of competitor proteins [core histones, (H3-H4)2, and BSA] to SET/TAF-Iβ/INHAT-bound histone–agarose. (E) Histone chaperone activity of SET/TAF-Iβ/INHATΔC. Circular plasmid DNA (lane 1) was relaxed by topoisomerase I and then incubated with (lanes 2–4) or without (lanes 5–7) core histones plus SET/TAF-Iβ/INHAT WT (lanes 3 and 6), SET/TAF-Iβ/INHATΔC (lanes 4 and 7), or no protein (lanes 2 and 5). Under these conditions, a small amount of supercoiled DNA formed in the absence of SET/TAF-Iβ/INHAT (lane 2). R, relaxed; S, supercoiled.
Fig. 2.
Fig. 2.
Structure of SET/TAF-Iβ/INHATΔC. (A) Amino acid sequences of SET/TAF-Iβ/INHAT and NAP-1. The α-helices (yellow) and β-strands (green) in the sequences are indicated. Residues modified by site-directed mutagenesis (see Fig. 4 A–C and SI Table 2) are red, and those with no observable electron density are not capitalized. Residues in the acidic stretch are gray. (B) Overall structure of the SET/TAF-Iβ/INHATΔC dimer, with the pseudo twofold axis highlighted in red. (C) The structure of the earmuff domain.
Fig. 3.
Fig. 3.
Comparison of the structures of SET/TAF-Iβ/INHAT and NAP-1. (A) Bottom (Left) and front (Right) views of the superimposed structures of NAP-1 (green) and SET/TAF-Iβ/INHAT (red). The yellow helices indicated by the arrows are the extra helix of NAP-1 that is inserted between the backbone helix and the earmuff domain (domain II). The rotation angle for the superimposition of the earmuff domains of NAP-1 and SET/TAF-Iβ/INHAT is given in the front view. Helices in the earmuff domain are not shown for clarity. (B) The superimposition of the earmuff domains of NAP-1 (green and blue) and SET/TAF-Iβ/INHAT (red). The extra residues that are unique to the NAP-1 structure are shown in blue. The corresponding residues of fragment A could not be modeled in the present study because of disordering. Fragment B is a long insertion of NAP-1 (see Fig. 2A).
Fig. 4.
Fig. 4.
Mapping of SET/TAF-Iβ/INHAT activities. (A–C) Location of triple mutated residue sets as observed from the top view (A), side view (B), and bottom view (C) of SET/TAF-Iβ/INHAT. For detailed information, see SI Table 2. (D) SDS/PAGE of the SET/TAF-Iβ/INHAT WT and mutant proteins and staining with Coomassie brilliant blue. For full gel information, see SI Fig. 7. (E) Histone chaperone activities of the SET/TAF-Iβ/INHAT WT and mutant proteins. The estimated relative net activity of each mutant is as follows: WT, 100; A, 103; B, 94; C, 98; D, 87; E, 91; F, 97; G, 99; H, 97; I, 104; J, 98; K, 98; L, 93; M, 88; N, 38; O, 36; P, 67; Q, 64; R, 95 (see Materials and Methods). (F and G) Histone-binding (F) and dsDNA-binding (G) activities of the SET/TAF-Iβ/INHAT WT and mutant proteins. The bound proteins were detected by immunoblot analysis using anti-His antibodies. Mutants N, O, and P demonstrated impaired binding activities. The estimated relative histone binding activity of each mutant is as follows: WT, 100; A, 98; B, 101; C, 91; D, 127; E, 98; F, 108; G, 103; H, 101; I, 108; J, 106; K, 96; L, 81; M, 100; N, 0; O, 0; P, 5; Q, 70; R, 122. The estimated relative DNA binding is as follows: WT, 100; A, 71; B, 89; C, 114; D, 121; E, 123; F, 117; G, 124; H, 138; I, 88; J, 77; K, 79; L, 73; M, 64; N, 5; O, 7; P, 0; Q, 29; R, 59 (see Materials and Methods).
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