Chemical and structural biology of protein lysine deacetylases

Abstract

Histone acetylation is a reversible posttranslational modification that plays a fundamental role in regulating eukaryotic gene expression and chromatin structure/function. Key enzymes for removing acetyl groups from histones are metal (zinc)-dependent and NAD⁺-dependent histone deacetylases (HDACs). The molecular function of HDACs have been extensively characterized by various approaches including chemical, molecular, and structural biology, which demonstrated that HDACs regulate cell proliferation, differentiation, and metabolic homeostasis, and that their alterations are deeply involved in various human disorders including cancer. Notably, drug discovery efforts have achieved success in developing HDAC-targeting therapeutics for treatment of several cancers. However, recent advancements in proteomics technology have revealed much broader aspects of HDACs beyond gene expression control. Not only histones but also a large number of cellular proteins are subject to acetylation by histone acetyltransferases (HATs) and deacetylation by HDACs. Furthermore, some of their structures can flexibly accept and hydrolyze other acyl groups on protein lysine residues. This review mainly focuses on structural aspects of HDAC enzymatic activity regulated by interaction with substrates, co-factors, small molecule inhibitors, and activators.

Keywords: acetylation; acylation; cancer; chromatin; epigenetics; sirtuin.

Figure 1.

Number of publications regarding histone deacetylases per year. Data were obtained from the PubMed (https://www.ncbi.nlm.nih.gov/pubmed) based on the keywords of “histone deacetylase”, “histone deacetylases”, “HDAC”, and “HDACs”. The years of identification of TSA and TPX as HDAC inhibitors are indicated.

Figure 2.

Structures of small molecule modulators of lysine deacetylases. (A) Structures of HDAC inhibitors. HDAC inhibitors are classified according to the structural signatures. (B) Structures of sirtuin inhibitors. (C) Structures of sirtuin activators. Structures of SIRT1460, STR1720 and STR2183 are shown as representatives of STACs.

Figure 3.

Chromatin status regulated by histone acetylation. Histone acetyltransferases (HATs) are recruited by transcriptional regulators such as transcription factors and co-activators to the particular genetic loci, which form potentially active chromatin. On the other hand, histone deacetylases (HDACs) are recruited by transcriptional repressors or co-repressors and are associated with transcriptionally inactive chromatin. Specific HDAC inhibitors such as TSA and TPX A inhibit HDACs, leading to histone hyperacetylation in vivo.

Figure 4.

Crystal structures and catalytic mechanisms of HDAC proteins. (A) HDLP (PDB id: 1C3R). TSA and zinc ion are represented as space-filled spheres, in which C, N, O, and Zn atoms are colored in yellow, blue, red, and grey, respectively. Close-up view of the active site is shown in the right panel. Two asparagine residues and histidine (underlined) coordinate the active site Zn²⁺ ion. (B) Arginase (1RLA). (C) The chemical structure of TSA, consisting of a cap, a spacer, and a zinc-binding functional group. (D) HDAC8 (1T64). K⁺ ion (Na⁺ ion in this structure due to the crystallization conditions) is colored in khaki. (E) Catalytic mechanism of class I HDACs. The catalytic histidine residue facilitates the nucleophilic attack at the substrate carbonyl by activating a water molecule (blue arrow). Hydrogen bond interactions are drawn in dotted lines. (F) HDAC3 in complex with inositol-tetraphosphate and DAD (4A69). The inositol phosphate is represented as space-filled spheres, in which C, O, and P atoms are depicted in yellow, red, and orange, respectively. The DAD is colored in green.

Figure 5.

Crystal structures and catalytic mechanism of sirtuin family proteins. (A) SirTm2 in complex with acetyl lysine peptide and NAD⁺ (PDB id: 2H4F), (B) SIRT2 in complex with acyl-ADP-ribose, an intermediate of the reaction between an acyl-peptide and NAD⁺ (4Y6Q), and (C) SIRT5 in complex with N-succinyl lysine peptide (3RIY). Active site histidine and substrates are represented as stick models, in which yellow, blue, red, and orange represent C, N, O, and P atoms, respectively. C atoms of NAD⁺ and acyl-ADP-ribose are in purple for clarity. Zn²⁺ ions are represented as space-filled spheres in grey. Close-up views of the active sites are shown in lower panels. (D) Catalytic mechanism of sirtuin family proteins. Acyl/acetyl-lysine residue is deacyl/deacetylated by nucleophilic addition of the acetamide oxygen to the C1′ position of the nicotinamide ribose. Nicotinamide and a deacylated/deacetylated peptide and a 2′-O-acyl/acetyl-ADP-ribose are final reaction products.

Figure 6.

A unique mode of action of FK228. FK228 is activated by cellular reducing activity in cells, forming thiol groups from its intramolecular disulfide bond. One the thiols may act as a zinc-binding ligand to inhibit HDAC.

Figure 7.

Regulation of cytoplasmic proteins by reversible acetylation. (A) Identification of HDAC6 as a tubulin deacetylase. By using the difference in target enzyme specificity between TSA and TPX, HDAC6 was identified as a tubulin deacetylase. TSA treatment increases α-tubulin acetylation, while TPX, which cannot inhibit HDAC6, fails to increase the α-tubulin acetylation. (B) Regulation of cell motility by cortactin acetylation and interaction with Keap1. Keap1, known as a negative regulator of the transcription factor Nrf2, was identified as a novel partner of cortactin, which regulates nuclear-cytoplasmic transport of cortactin. In addition, cortactin interaction with Keap1 is required for cortical localization of cortactin. Acetylation of cortactin inhibits the interaction with Keap1, thereby controlling cell migration. Cortactin is acetylated by acetyltransferase activity of the nuclear CBP, which is also by regulated by deacetylase activity of SIRT1 in the nucleus. Upon growth stimulation, cortactin is deacetylated by two cytoplasmic deacetylases, HDAC6 and SIRT2, which promotes cortical translocation and cell migration.

Figure 8.

Summary of lysine N-acyl modifications and related enzymes.