Structural Studies of Human Histone Deacetylase 8 and Its Site-Specific Variants Complexed with Substrate and Inhibitors^,

Abstract

Metal-dependent histone deacetylases (HDACs) require Zn2+ or Fe2+ to regulate the acetylation of lysine residues in histones and other proteins in eukaryotic cells. Isozyme HDAC8 is perhaps the archetypical member of the class I HDAC family and serves as a paradigm for studying structure-function relationships. Here, we report the structures of HDAC8 complexes with trichostatin A and 3-(1-methyl-4-phenylacetyl-1H-2-pyrrolyl)-N -hydroxy-2-propenamide (APHA) in a new crystal form. The structure of the APHA complex reveals that the hydroxamate C=O group accepts a hydrogen bond from Y306 but does not coordinate to Zn2+ with favorable geometry, perhaps due to the constraints of its extended π system. Additionally, since APHA binds to only 2 of the 3 protein molecules in the asymmetric unit of this complex, the structure of the third monomer represents the first structure of HDAC8 in the unliganded state. Comparison of unliganded and liganded structures illustrates ligand-induced conformational changes in the L2 loop that likely accompany substrate binding and catalysis. Furthermore, these structures, along with those of the D101N, D101E, D101A, and D101L variants, support the proposal that D101 is critical for the function of the L2 loop. However, amino acid substitutions for D101 can also trigger conformational changes of Y111 and W141 that perturb the substrate binding site. Finally, the structure of H143A HDAC8 complexed with an intact acetylated tetrapeptide substrate molecule confirms the importance of D101 for substrate binding and reveals how Y306 and the active site zinc ion together bind and activate the scissile amide linkage of acetyllysine.

Activation and Inhibition of Histone Deacetylase 8 by Monovalent Cations

“…Similar slow conformational transitions have been reported for other proteins (24,25), and conformational plasticity has been linked to allosteric activation by MVCs (26). Crystal structures of HDAC8 complexes with different inhibitors or substrates demonstrate that the protein displays significant structural plasticity (11,12,27). Because both MVC sites are occupied in all of the published crystal structures of HDAC8, the nature of this proposed conformational change upon MVC binding to site 2 is not known but would be expected to influence the positioning of active site residues.…”

Inhibitors of Human Histone Deacetylase: Synthesis and Enzyme Assay of Hydroxamates with Piperazine Linker

“…Possibly a functional group with a hydrogen-bonding capability with TYR100 in the cap aryl region may increase the interaction and, thereby, increase the potency. The observation is consistent with the recent report of the importance of ASP101 of HDAC8 in establishing hydrogen-bond interac- tion with the backbone NH of the substrate and different classes of HDAC inhibitors [21]. Interestingly, none of the N-hydroxyacetamide derivatives was found to establish a hydrogen-bonding interaction with ASP101, and, owing to this, the cap aryl group pointed away from TYR100 (Fig.…”

Histone deacetylases: structural determinants of inhibitor selectivity

“…To date, 39 crystal structures of human HDACs (isoforms 1, 2, 3, 4, 7, and 8) and eight for HDAC homologs from bacteria (HDLP and HDAH) have been solved [19,40,[43][44][45][46][47][48][49][50][51][52][53][54][55][56][57][58][59][60] (Tables 2 and 3). …”

“…Also, the histidine residue of the partially solvent-exposed charge relay system seems to have a crucial role in catalysis, because the H151A mutation in Rpd3 and H143A in HDAC8 (corresponding to His670 in HDAC7, Fig. 2d) make the enzyme completely deficient in HDAC activity [56,61]; in addition, mutation of the same residue in HDAC1 (H141A) [62] drastically reduces deacetylase activity. Another residue involved in catalysis is a tyrosine (Y308 in HDAC2 as shown in Fig.…”