HDAC8 substrate selectivity is determined by long- and short-range interactions leading to enhanced reactivity for full-length histone substrates compared with peptides

Abstract

Histone deacetylases (HDACs) catalyze deacetylation of acetyl-lysine residues within proteins. To date, HDAC substrate specificity and selectivity have been largely estimated using peptide substrates. However, it is unclear whether peptide substrates accurately reflect the substrate selectivity of HDAC8 toward full-length proteins. Here, we compare HDAC8 substrate selectivity in the context of peptides, full-length proteins, and protein-nucleic acid complexes. We demonstrate that HDAC8 catalyzes deacetylation of tetrameric histone (H3/H4) substrates with catalytic efficiencies that are 40-300-fold higher than those for corresponding peptide substrates. Thus, we conclude that additional contacts with protein substrates enhance catalytic efficiency. However, the catalytic efficiency decreases for larger multiprotein complexes. These differences in HDAC8 substrate selectivity for peptides and full-length proteins suggest that HDAC8 substrate preference is based on a combination of short- and long-range interactions. In summary, this work presents detailed kinetics for HDAC8-catalyzed deacetylation of singly-acetylated, full-length protein substrates, revealing that HDAC8 substrate selectivity is determined by multiple factors. These insights provide a foundation for understanding recognition of full-length proteins by HDACs.

Keywords: enzyme kinetics; enzyme turnover; histone; histone acetylation; histone deacetylase (HDAC); non-natural amino acid incorporation; nucleosome; peptides; protein complex.

Figure 1.

Shown is the structure of histone H3/H4 tetramer with highlighted acetylation sites. Shown is the structure of histone H3/H4 tetramer (62) with boxes around the sites that were acetylated. H3 is shown in blue and H4 in yellow. H3 residues 1–20 are shown in an extended conformation as they have no discrete fold within the crystal structure. The structure was generated from Protein Data Bank code 1AOI using VMD.

Figure 2.

Single turnover deacetylation of singly-acetylated H3/H4 tetramers. A, sample data from a deacetylation reaction (7 μm HDAC8 and 0.5 μm H3K9ac/H4 tetramer (1 μm acetyl-lysine)) measured using mass spectrometry. The time-dependent decrease in acetylated protein is best described by a single exponential. B, dependence of apparent deacetylation rate constant of H3K9ac/H4 on the concentration of HDAC8. The k_obs average of 0.021 ± 0.001 s⁻¹ shows little dependence on the [HDAC8]. Three separate hyperbolic fits are shown that bracket potential K_½ values: K_½ = 0.5 μm (dotted line); K_½ = 1.0 μm (dashed line); and K_½ = 1.5 μm (solid line). These fits demonstrate that the K_½ is <1.5 μm and k_max/K_½ is >17,000 m⁻¹ s⁻¹. The data points are from multiple measurements in a single reaction at each HDAC8 concentration. C, dependence of the deacetylation rate constant for H3K14ac/H4 on the concentration of HDAC8. The data points are from multiple measurements in a single reaction at each HDAC8 concentration. A hyperbolic fit indicates that the k_max/K_½ is 2,500 ± 70 m⁻¹ s⁻¹ with an estimated value for k_max of 0.06 s⁻¹. D, dependence of the deacetylation rate constant for H3K56ac/H4 on the concentration of HDAC8. The data points are from multiple measurements in a single reaction at each HDAC8 concentration. A linear fit indicates that the k_max/K_½ is 4,000 ± 600 m⁻¹ s⁻¹.

Figure 3.

Single turnover deacetylation of singly-acetylated H3 octamers. A, dependence of the apparent deacetylation rate constant of H3K9ac octamer on the concentration of HDAC8. Data points are from multiple measurements in a single reaction at each HDAC8 concentration, and error bars on k_obs values represent errors calculated from the exponential fits. A linear fit of the data indicates that the k_max/K_½ is 3,700 ± 100 m⁻¹ s⁻¹. B, dependence of the apparent deacetylation rate constant of H3K14ac octamer on the concentration of HDAC8. The data points are from multiple measurements in a single reaction at each HDAC8 concentration. A hyperbolic fit indicates that the k_max/K_½ is 1,000 ± 200 m⁻¹ s⁻¹ with a k_max value of 0.03 ± 0.02 s⁻¹.

Figure 4.

Single turnover deacetylation of singly-acetylated H3 nucleosome. The initial rate of progress curves for deacetylation of H3K9ac nucleosome catalyzed by 0–7.5 μm HDAC8 was fit linearly, and the rate constant was calculated assuming 100% deacetylated product. The data points are from multiple measurements in a single reaction at each HDAC8 concentration, and error bars represent errors calculated from the initial rate fits. A linear fit of the data indicates that k_max/K_½ is 28 ± 3 m⁻¹ s⁻¹.