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Structural highlights
HDAC8 was the first among the HDAC isozymes whose crystal structure became available. The image shows the ribbon structure of HDAC8 bound with a canonical hydroxamate inhibitor, SAHA (Suberoylanilide Hydroxamic Acid) [pdb 1T69]. HDAC8 contains a single α/β-deacetylase domain consisting of 13 α-helices and an eight-stranded parallel β-sheet. Multiple loops, which emanate from the protein core, play a significant role in maintaining the appropriate geometry of the catalytic pocket. The α/β-fold of the above kind was first observed in a metalloenzyme, Arginase. The active site of HDAC8 contains a tubular cavity leading to catalytic machinery at the end. The catalytic machinery is comprised of a Zn2+ ion penta-coordinated with a square pyramidal geometry. The residues His 180, Asp 267 and Asp 176 occupy the three co-ordination sites, whereas the hydroxamate moiety of SAHA occupies the remaining two sites. In addition, the carbonyl oxygen of the hydroxamate moiety forms a hydrogen bond with Tyr 306. In the absence of any ligand (substrate/inhibitors), two water molecules are bound to the Zn2+ ion. The linker domain of the inhibitor interacts with the residues: Phe 152, Phe 208, His 180, Gly 151, and Met 274, which forms a hydrophobic tunnel. Notably, the above residues involved in the inhibitor/substrate binding, as well as the catalysis, have been found to be conserved during the course of evolution among class I HDACs. A single Zinc-bound water molecule present in the HDAC8-substrate complex serves as a nucleophile in the deacetylation reaction. In addition, it makes hydrogen bonding with the N2 atom of imidazole of His 142 and His 143, which reportedly form a charge-relay system, respectively, with Asp 176 and Asp 183. The charge-relay system enhances the basicity of the N2 of the imidazole and has been reported previously in serine proteases [57]. Notably, the carbonyl oxygen of the acetyl-lysine substrate forms a hydrogen bond with the hydroxyl moiety of Y306.
Aside from the catalytic Zn2+ ion, the enzyme activity of HDAC8 is dependent on the presence of the monovalent ion, K+ [60]. The crystal structure of HDAC8 shows the presence of two binding sites for K+ [58]. The first K+ binding site (K1) is located in the vicinity of the enzyme catalytic machinery, and it is hexacoordinated (octahedral geometry) with His 180 (carbonyl oxygen of the main chain), Asp 176 (oxygen atom of the main chain and side chain), Leu 200 (carbonyl oxygen of the main chain), and Ser 199 (O). Notably, His 180 and Asp 176 are the common residues coordinated with both the catalytic Zn2+ as well as the K+ ion. The second binding site for K+ ion (K2) is located 15Å away from the catalytic Zn2+ ion. It is hexacoordinated (octahedral geometry) with F189, T192, V195, Y225 as well as two water molecule.
The crystal structure of HDAC8-substrate complex (pdb 2V5W) elucidates the role of an aspartate residue (D101) in the substrate binding [59]. Asp 101 resides on the L2 loop and its carboxylate moiety makes two consecutive hydrogen bonds with the backbone of the p53-derived deacetylated peptide substrate as shown in Figure 1.3. Mutation of Asp 101 to Ala abolishes the HDAC8 catalytic activity, signifying its role in substrate binding. More importantly, the Asp residue has been found to be strictly conserved among different HDAC isozymes, further emphasizing its importance in the substrate binding.
Based on the crystallographic studies, a mechanism of the HDAC8 catalyzed reaction has been proposed. The zinc ion plays a pivotal role in the entire catalytic process. It reduces the entropy by bringing the water and the acetyl-lysine substrate together to initiate the deacetylation reaction. Binding of the substrate to Zn2+ polarizes its carbonyl group thereby increasing its electrophilicity. In addition, the pKa of the Zn2+ bound water molecule is lowered, making it a stronger nucleophile. The deacetylation reaction starts with a nucleophilic attack of the catalytic water on the carbonyl carbon of the acetyl-lysine, producing a tetrahedral oxyanion intermediate that is stabilized via an electrostatic interaction with the Zn2+ ion as well as a hydrogen bonding with the hydroxyl group of Tyr 306. The collapse of the tetrahedral intermediate is mediated via the transfer of a proton from His 142, leading to the production of an acetate ion and a deacetylated lysine.
The release of the acetate ion generated during the HDAC8 catalysis is mediated via an acetate release channel (exit tunnel), which is located adjacent to the active site pocket.
HDAC8 has several intriguing features which distinguish it from other HDAC isozymes. It lacks the 50-111 AAs segment extending beyond the C-terminal of the catalytic domain which is utilized to recruit other co-repressor/transcription factors [57]. The crystallographic studies of HDAC8 with the structurally diverse hydroxamate inhibitors, namely, TSA, SAHA, M-334 and CRA-A, reveal an inherent malleability of its active site pocket, which is primarily due to the presence of the L1 loop (S30-K38) [57]. Moreover, the phosphorylation of Ser 39 by PKA has been reported to reduce the rate of the HDAC8 enzyme catalyzed reaction, presumably by slowing down the release of acetate through the release channel mediated via an electrostatic repulsion [58].
The crystal structure of HDAC8 bound with a depsipeptide inhibitor, Largazole (pdb 3RQD), was solved [62]. The inhibitor is a pro-drug which produces a thiol moiety upon hydrolysis of the thioester linkage. The structure observed here was similar to the HDAC8-SAHA complex with the following salient differences. The thiolate ion serves as a monodentate ligand for the Zn2+ ion as opposed to SAHA, which is bidendate. The zinc ion coordination geometry is tetrahedral in case of HDAC8-largazole thiol complex, which is unique among all the crystal structures of HDAC isozymes known so far. Additionally, HDAC8 was required to undergo relatively larger conformational changes in the L1 and L2 loop region to accommodate the bulky and rigid inhibitor, largazole.
Additionally, it was possible to obtain the crystal of the apo form of HDAC8, which was free from any ligand. A comparison of the ligand-bound (holoenzyme) vs. ligand -free (apoenzyme) forms of HDAC8 showed that the L2 loop becomes ordered upon binding to the ligand, APHA, 3-(1-methyl-4-phenylacetyl-1H-2-pyrrolyl)-N-hydroxy-2-propenamide [64].
