Danilenko N et al. (2019), Histone chaperone exploits intrinsic disorder t...

XB-ART-56529

Nat Commun 2019 Aug 06;101:3435. doi: 10.1038/s41467-019-11410-7.

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Histone chaperone exploits intrinsic disorder to switch acetylation specificity.

Danilenko N , Lercher L , Kirkpatrick J , Gabel F , Codutti L , Carlomagno T .

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Histones, the principal protein components of chromatin, contain long disordered sequences, which are extensively post-translationally modified. Although histone chaperones are known to control both the activity and specificity of histone-modifying enzymes, the mechanisms promoting modification of highly disordered substrates, such as lysine-acetylation within the N-terminal tail of histone H3, are not understood. Here, to understand how histone chaperones Asf1 and Vps75 together promote H3 K9-acetylation, we establish the solution structural model of the acetyltransferase Rtt109 in complex with Asf1 and Vps75 and the histone dimer H3:H4. We show that Vps75 promotes K9-acetylation by engaging the H3 N-terminal tail in fuzzy electrostatic interactions with its disordered C-terminal domain, thereby confining the H3 tail to a wide central cavity faced by the Rtt109 active site. These fuzzy interactions between disordered domains achieve localization of lysine residues in the H3 tail to the catalytic site with minimal loss of entropy, and may represent a common mechanism of enzymatic reactions involving highly disordered substrates.

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Species referenced: Xenopus
Genes referenced: asf1a fuz

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	Fig. 1. Vps752 forms a doughnut-shaped complex with Asf1, H3:H4 and Rtt109. a Overlay of 1H-13C HMQC spectra of ILV methyl-labeled Vps752 in isolation, with Rtt109 and in the Asf1â€“H3:H4â€“Rtt109â€“Vps752 complex. In Vps752, Vps75(A) and Vps75(B) have identical chemical shifts (left). In complex with Rtt109 (middle), only Vps75(B) displays CSPs. Upon further addition of Asf1â€“H3:H4 (right), the Vps75(B) peaks do not shift further, while Vps75(A) displays noticeable CSPs. Thus, Vps75(B) recruits Rtt109, while Vps75(A) binds Asf1â€“H3:H4. Spectra were recorded on samples of 60â€‰Î¼M Vps752 in 50â€‰mM sodium citrate pH 6.5, 150â€‰mM NaCl, 5â€‰mM BME at 850â€‰MHz and 298â€‰K. b The Asf1â€“H3:H4â€“Rtt109â€“Vps752 complex adopts a doughnut-like shape with a central cavity of ~25â€‰Ã… width. The Rtt109 C-terminal tail (Rtt109419â€“433) is shown bound to Asf1, as described in Lercher et al.34 (PDB entry 6f0y). A flat ribbon indicates the portion of the Rtt109 C-terminal tail for which no structural information is available. The disordered Vps75 and H3 tails are not shown. c Electrostatic surface representation of the inner part of the complex. D225 and G231 are the last structured amino acids of Vps752; L60 is the first amino acid of the H3 core; the Vps75-206EE207 dyad is at the center of an acidic patch
	Fig. 2. K9-acetylation does not depend on the H4 C-terminal region. a The interface of H3, Rtt109 and Vps75(A) displays a network of hydrogen bonds (dashed lines) and electrostatic contacts. b Interface between the H4 C-terminal region and Rtt109. c, d Interface of H3:H4 and Vps75(A): hydrophobic contacts involve H4-L22, H3-R69 and L65, Vps75-F77, Q64, A219 and the backbone of stretch 70â74; electrostatic contacts involve Vps75-K170 and Q174 and H3-S85. e, f Time-courses (left) of K56- and K9-acetylation quantified by dot-blot assays (right). The H4Â 95â102 stretch is important for K56ac (e) but not for K9ac (f). Experimental conditions: 0.2âÎ¼M Rtt109âVps752, 0.2âÎ¼M Asf1âH3:H4, 2âÎ¼M Ac-CoA, 10âmM HEPES pH 8.0, 100âmM NaCl. Because the control reaction with 0.2âÎ¼M Asf1âH3:H4 (white) did not give signal above the background, an additional negative control with 6âÎ¼M Asf1âH3:H4 (column âcâ) was used for normalization and comparison of the experimental repeats. The data were averaged over four experiments; the error bars are the standard errors of the means. Source data are provided as a Source Data file
	Fig. 3. The Vps75 acidic patch keeps K56 away from the Rtt109 catalytic pocket. a Final snapshot of the 100-ns MD trajectory of Asf1âH335â135:H4âRtt109âVps7521â225, starting from the structure of Fig.Â 1. The stretch H335â59 was initially built in an extended conformation (Supplementary Fig.Â 8). During the MD run, stable H-bonds were formed between H3-R52 and Vps75(A)-E207 and between H3-R53 and Vps75(B)-E207; these contacts are incompatible with H3-K56 binding in the Rtt109 catalytic center. b Analysis of the H-bonds during the MD run. c, d Time-courses (left) of K56- and K9-acetylation quantified by dot-blot assays (right). The assays and data-analysis were done as described in the legend to Fig.Â 2. Source data are provided as a Source Data file
	Fig. 4. The Vps75 CTAD interacts with both the H3 core and tail. a Comparison of the 1H-13C HMQC spectra of ILV methyl-labeled H3:H4 in the Asf1â€“H3:H4 complex and upon addition of either 1.1 equivalents of Vps752 (left) or Vps7521â€“225 (right). The corresponding CSPs mapped on the Asf1â€“H3:H4 structure (PDB entry 2hue) are shown in the bottom-left and bottom-middle panel, respectively. The CSPs obtained after addition of 1.1 equivalents of Rtt109â€“Vps752 to Asf1â€“H3:H4 (bottom-right panel) are very similar to those obtained with Vps752 and demonstrate that the Vps75 CTAD interacts with the DNA-binding surface of H3:H4 in both Asf1â€“H3:H4â€“Vps752 and Asf1â€“H3:H4â€“Rtt109â€“Vps752. b Top, 1H-15N HSQC spectra of H3 in the H3:H4 dimer, in the Asf1â€“H3:H4 complex and upon further addition of Vps752, Vps7521â€“225 or Rtt109â€“Vps752. Bottom, the corresponding CSPs plot. In Asf1â€“H3:H4, the H3 tail interacts with Asf1, as reported by Lercher et al.34. In the presence of Vps7521â€“225, the H3 tail is released from Asf1 and recovers the CSs of the H3:H4 dimer; in the presence of Vps752, the H3 tail peaks move to new positions. The NMR experimental conditions were as in Fig.Â 1. Source data are provided as a Source Data file
	Fig. 5. The disordered Vps75 CTAD guides substrate specificity. a The SANS-derived ab initio envelopes of Asf1â€“H3:H4â€“Rtt109â€“Vps7521â€“225 (middle) and Asf1â€“H335â€“135:H4â€“Rtt109â€“Vps7521â€“225 (right) display a central empty cavity, which is filled by scattering units in the complex containing full-length proteins (left). Therefore the H3 tail does not enter the cavity in the absence of the Vps75 CTAD. b Overlay of 1H-15N spectra of Vps752 in isolation and as part of either Asf1â€“H3:H4â€“Rtt109â€“Vps752 or Asf1â€“H329â€“135:H4â€“Rtt109â€“Vps752. Only signals of the Vps75 CTAD are visible. In Asf1â€“H3:H4â€“Rtt109â€“Vps752, the CTADs of Vps75(A) and Vps75(B) display different chemical shifts: the peaks of one of the two CTADs remain in the same positions as those from Vps752, while those of the other CTAD move to new positions. The peaks of residues 234â€“246 move less far in Asf1â€“H335â€“135:H4â€“Rtt109â€“Vps752 (CSP plot, bottom). c TALOS-N CS analysis of Vps75226â€“264 in the context of full-length Vps752 shows that the Vps75 tail is disordered. The same narrow CS dispersion is observed in the full complex (Supplementary Fig.Â 9). d, e Time-courses (left) of K56- (d) and K9-acetylation (e) quantified by dot-blot assays (right). The assays and data-analysis were done as described in the legend to Fig.Â 2. Source data are provided as a Source Data file
	Fig. 6. Fuzzy electrostatic interactions promote acetylation of lysine residues in the H3 tail. Left, the mechanism of chaperoning H3-K56 to the Rtt109 catalytic pocket is based on well-known enzyme-recruitment and substrate-presentation processes. Right, the mechanism by which Vps75 chaperones lysine residues in the H3 tail to the Rtt109 catalytic pocket differs from the canonical substrate-presentation process and includes confinement of the H3 tail in the proximity of the Rtt109 catalytic pocket via fuzzy electrostatic interactions occurring between two disordered protein domains, the Vps75 CTAD and the H3 tail.

References [+] :

Abshiru, Chaperone-mediated acetylation of histones by Rtt109 identified by quantitative proteomics. 2013, Pubmed