Phosphorylation of Histone Deacetylase 8: Structural and Mechanistic Analysis of the Phosphomimetic S39E Mutant

Abstract

Histone deacetylase (HDAC) enzymes that catalyze removal of acetyl-lysine post-translational modifications are frequently post-translationally modified. HDAC8 is phosphorylated within the deacetylase domain at conserved residue serine 39, which leads to decreased catalytic activity. HDAC8 phosphorylation at S39 is unique in its location and function and may represent a novel mode of deacetylation regulation. To better understand the impact of phosphorylation of HDAC8 on enzyme structure and function, we performed crystallographic, kinetic, and molecular dynamics studies of the S39E HDAC8 phosphomimetic mutant. This mutation decreases the level of deacetylation of peptides derived from acetylated nuclear and cytoplasmic proteins. However, the magnitude of the effect depends on the peptide sequence and the identity of the active site metal ion [Zn(II) vs Fe(II)], with the value of k_cat/K_M for the mutant decreasing 9- to >200-fold compared to that of wild-type HDAC8. Furthermore, the dissociation rate constant of the active site metal ion increases by ∼10-fold. S39E HDAC8 was crystallized in complex with the inhibitor Droxinostat, revealing that phosphorylation of S39, as mimicked by the glutamate side chain, perturbs local structure through distortion of the L1 loop. Molecular dynamics simulations of both S39E and phosphorylated S39 HDAC8 demonstrate that the perturbation of the L1 loop likely occurs because of the lost hydrogen bond between D29 and S39. Furthermore, the S39 perturbation causes structural changes that propagate through the protein scaffolding to influence function in the active site. These data demonstrate that phosphorylation plays an important regulatory role for HDAC8 by affecting ligand binding, catalytic efficiency, and substrate selectivity.

Figure 1.. Structure of HDAC8.

Crystal structure of human, wild-type HDAC8 (grey, PDB ID 2V5W) bound to a peptide substrate derived from p53 (cyan). Loops L1 and L2 are shown in green and the active site zinc(II) ion is shown in red. Indicated are helices α1 and α2 and residues S39, R37, K33, D101, G303 and G305.

Figure 2.. Structural comparison of S39E and wild type HDAC8.

A. Stereoview superimposition of the S39E HDAC8-Droxinostat complex (monomer A: C = wheat, N = blue, O = red, Zn²⁺ = magenta sphere) and the wild-type HDAC8-M344 complex (PDB 1T67, color-coded as above except C = light blue). In the wild-type structure, S39 donates a hydrogen bond (black dashed line) to D29. Upon substitution to a glutamate (simulated omit map contoured at 4.0σ showing the E39 side chain), this interaction is not conserved and causes local rearrangement. The L1 loop adopts a different conformation as highlighted in red and blue for the S39E HDAC8-Droxinostat complex and the wild-type HDAC8-M344 complex, respectively. B. Stereoview simulated annealing omit map of Droxinostat bound in the active site of S39E HDAC8 (monomer A, contoured at 3.0σ). Atomic color codes are as follows: C = wheat (protein, monomer A), or green (inhibitor), N = blue, O = red, Zn²⁺ = magenta sphere. Metal coordination and selected hydrogen bond interactions are shown as solid black or dashed black lines, respectively. As in A, the L1 loop of S39E HDAC8 is highlighted in red.

Figure 3.. Deacetylation of LARP1 peptide by S39E and wild-type HDAC8.

Representative peptide assay data. Dependence of the initial rates of deacetylation of the LARP1 peptide (LGK(ac)FRR) on the substrate concentration catalyzed by Zn(II)-constituted S39E (closed blue circles, right y-axis) and wild-type HDAC8 (open blue circles, left y-axis) measured using the acetate assay. Enzyme concentration was 1 μM and substrate concentration was 10–800 μM. The data are a combination of two experiments (Zn(II)-S39E), or one experiment (Zn(II)-WT), and the Michaelis-Menten equation (Equation 1) was fit to the data using global regression analysis (GraphPad Prism). Standard errors were determined from the fits.

Figure 4.. Comparison of S39E and wild-type HDAC8 deacetylation of peptides.

A. Catalytic efficiencies, k_cat/K_M, of wild-type and S39E HDAC8-catalyzed deacetyation of peptides listed in Table 2 as measured by the Fluor de Lys assay (FdL-HDAC8 and FdL-Sirt1 peptides) and the acetate assay (remaining peptides). B. fold change in catalytic efficiency of S39E HDAC8 compared to wild type for peptides. For all three graphs, peptides are ordered from most to least active with wild-type HDAC8. Error bars are shown in same colors as columns. Substrate names on X-axis correspond to peptides listed in Table 2.

Figure 5.. Zinc(II)- and iron(II)-constituted S39E and wild-type HDAC8 catalyzed deacetylation of fluorescently-labeled Fluor de Lys HDAC8 test substrate.

Dependence of the initial rates of deacetylation of the Fluor-de-Lys HDAC8 peptide substrate on substrate concentration catalyzed by A. Zn(II)-constituted S39E HDAC8 (closed blue circles) and Zn(II)-constituted WT HDAC8 (open blue circles) and B. Fe(II)-constituted S39E HDAC8 (closed red circles) and Fe(II)-constituted WT HDAC8 (open red circles). Enzyme concentration was 0.5–1 μM and substrate concentration was 10–1000 μM. The data are a combination of four experiments (Zn(II)-S39E), or one experiment (Zn(II)-WT, Fe(II)-S39E, Fe(II)-WT), and the Michaelis-Menten equation (Equation 1) was fit to the data using global regression analysis (GraphPad Prism).

Figure 6.. Metal ion dissociation rates for zinc(II) and iron(II)-constituted S39E HDAC8.

Initial rates for Zn(II)-constituted S39E HDAC8 (filled blue circles) and Fe(II)-constituted S39E HDAC8 (filled red circles) deacetylation activity as a function of time after addition of 1 mM EDTA, as measured using the Fluor-de-Lys assay. The fraction activity is determined by dividing the activity in EDTA by the activity of HDAC8 incubated in the absence of EDTA. The k_off values were calculated by fitting an exponential decay equation to data from replicates on different days using global regression analysis (GraphPad Prism).

Figure 7.. Simulations of wild-type HDAC8 binding to substrate.

Top panel (red box): The orientation of key residues in wild-type HDAC8 at A. the start, B. 51 ns, and C. 70 ns of the substrate binding simulation. S39 is solvent-exposed. Y306 bends at 90° toward K33, and the Hε of Y306 forms a hydrogen bond with the carbonyl of K33 (B). This opens the tunnel for substrate interaction with the active site, which is otherwise blocked by Y306. Yellow and purple spheres represent Zn²⁺ and K⁺ ions respectively. Center and bottom panels: Simulations of pS39 (blue box) and S39E (green box) bound to substrate. In the center panel (pS39 modeled on wild-type HDAC8), two snapshots (D. start and E. 400 ns) during the simulation demonstrate that the substrate is shifted in the active site between K33 and Y306 compared to wild-type HDAC8 (Figure 7A–C). Y306 interacts with substrate but does not interact with K33. The bottom panel (S39E HDAC8 with modeled substrate) is a representation at F. the start and G. 400 ns of the simulation of key residues in S39E HDAC8 and demonstrates that the enzyme behaves similarly to pS39-HDAC8. The L1 loop is altered, Y306 and K33 do not interact (unlike in wild-type HDAC8 (Figure 7B) where Y306 forms a hydrogen bond with the carbonyl oxygen of K33), and Y306 does not interact with substrate in this simulation. Substrate access to the active site is altered. Yellow and purple spheres represent Zn²⁺ and K⁺ ions respectively.

Figure 8.. Interaction between K36-D29-S39.

A. The wild-type (blue) and mutant HDAC8 (yellow) is illustrated. The S39-D29 interaction tethers loop 1 and maintains the orientation of K33 in the wild-type protein. S39E can interact with K36 but not with D29 and therefore K33 orientation is not maintained in the mutant. B-C. Distance plot showing the interaction between D29, K36 and S39 in (B) wild-type and (C) S39E mutant HDAC8. In wild type, S39 can interact with D29 directly while in S39E, mutant residue S39E and D29 are beyond interacting distance (green).