Structure, mechanism, and inhibition of the zinc-dependent histone deacetylases

Abstract

Zinc-dependent histone deacetylases (HDACs) regulate the biological function of histone and non-histone proteins through the hydrolysis of acetyllysine side chains to yield free lysine and acetate. Certain HDAC isozymes exhibit alternative catalytic activities, such as polyamine deacetylase or lysine fatty acid deacylase activity. To date, crystal structures have been reported for class I HDACs (1, 2, 3, and 8), class IIa HDACs (4 and 7), and class IIb HDACs (6 and 10). Conserved active site residues mediate the chemistry of substrate activation and hydrolysis in these isozymes through a metal-activated water molecule assisted by general base-general acid catalysis. Upregulated HDAC activity is observed in cancer and neurodegenerative disease, and four HDAC inhibitors are currently approved for use in cancer chemotherapy. Crystal structures of HDAC-inhibitor complexes guide the design of new inhibitors with high affinity and selectivity for specific HDAC isozymes implicated in human disease.

Figure 1:. Arginase-deacetylase family of proteins.

Unrooted phylogenetic tree indicates 12 clades: arginases, pseudo-arginases (ΨARG), formiminoglutamases (FIGase) and ureohydrolases, yeast Hos3 homologues, bacterial acetylpolyamine amidohydrolases (APAH), bacterial histone deacetylase-like amidohydrolases (HDAH), class II HDACs, class I HDACs, bacterial acetoin utilization proteins (AcuC), class IV HDACs, uncharacterized protein family UPF0489, and pseudo-deacetylases (ΨDAC). Individual proteins, species, and UniProt accession numbers are listed in Supplementary Table 3 of ref. [••13]. Reprinted from ref. [••13] (Creative Commons Attribution 4.0 International License).

Figure 2:. Proposed mechanism of acetyllysine hydrolysis catalyzed by HDAC8.

Enzymological measurements indicate that H143 serves as a single general base-general acid, while H142 remains in the positively charged imidazolium form and serves as an electrostatic catalyst. However, hydrogen bond differences for the tandem histidine pair in other HDAC isozymes may allow for dual general base-general acid function. For example, in HDAC6, H573 may serve as a general base, and H574 may serve as a general acid [••33].

Figure 3:. HDAC complexes with cyclic tetrapeptide inhibitors.

(a) Structure of the HDAC8-Trapoxin A complex determined at 1.24 Å resolution (PDB 5VI6). The α,β-epoxyketone side chain of L-Aoe binds as a gem-diolate, thereby mimicking the tetrahedral intermediate and its flanking transition states in catalysis. The flanking NH groups of L-Aoe donate hydrogen bonds to D101, a key determinant of isozyme-substrate recognition. Reproduced from ref [••51]. Copyright 2017 American Chemical Society. (b) Structure of the HDAC6-HC Toxin complex determined at 1.73 Å resolution (PDB 5EFJ). Here, too, the α,β-epoxyketone side chain binds as a gem-diolate. The backbone NH group of L-Aoe donates a hydrogen bond to S531, a key determinant of isozyme-substrate recognition. Reprinted from ref [••33].

Figure 4:. Substrate binding to HDACs.

(a) Superposition of substrate complexes with Y306F HDAC8 (blue, PDB 2V5W) and H143A HDAC8 (grey, PDB 3EWF) reveals that the scissile carbonyl of acetyllysine is activated by Zn²⁺ coordination and a hydrogen bond with Y306. The flanking NH groups of the acetyllysine substrate donate hydrogen bonds to D101. (b) Superposition of substrate complexes with Y745F HDAC6 (light green, PDB 5EFK) and H574A HDAC6 (dark green, PDB 5EFN) similarly reveals activation of the scissile substrate carbonyl by Zn²⁺ coordination and a hydrogen bond with Y745. Moreover, nucleophilic attack of Zn²⁺-bound water at the substrate carbonyl group occurs in H574A HDAC6 to result in a tetrahedral intermediate. The backbone NH group of acetyllysine donates a hydrogen bond to S531. (c) The trifluoromethylketone analogue of N⁸-acetylspermidine binds as a tetrahedral gem-diolate that mimics the tetrahedral intermediate in the active site of HDAC10 (PDB 5TD7). A 3₁₀ helix unique to this isozyme (magenta) sterically constricts the active site and confers specificity for acetylpolyamine substrates.

Figure 5:. HDAC inhibitors.

(a) Hydroxamate-based HDAC inhibitors currently approved for cancer chemotherapy. (b) The hydroxamate moiety is susceptible to degradation through the Lossen rearrangement, which generates an electrophilic isocyanate. (c) Romidepsin is currently approved for cancer chemotherapy and is activated by reduction of the disulfide linkage. The marine natural product Largazole is structurally similar to Romidepsin and is activated by thioester hydrolysis. (d) The crystal structure of the HDAC8-Largazole complex shows that the ionized thiolate side chain coordinates to Zn²⁺ and accepts a hydrogen bond from Y306 (PDB 3RQD).