Structure, mechanism, and inhibition of histone deacetylases and related metalloenzymes

Abstract

Metal-dependent histone deacetylases (HDACs) catalyze the hydrolysis of acetyl-L-lysine side chains in histone and nonhistone proteins to yield l-lysine and acetate. This chemistry plays a critical role in the regulation of numerous biological processes. Aberrant HDAC activity is implicated in various diseases, and HDACs are validated targets for drug design. Two HDAC inhibitors are currently approved for cancer chemotherapy, and other inhibitors are in clinical trials. To date, X-ray crystal structures are available for four human HDACs (2, 4, 7, and 8) and three HDAC-related deacetylases from bacteria (histone deacetylase-like protein (HDLP); histone deacetylase-like amidohydrolase (HDAH); acetylpolyamine amidohydrolase (APAH)). Structural comparisons among these enzymes reveal a conserved constellation of active site residues, suggesting a common mechanism for the metal-dependent hydrolysis of acetylated substrates. Structural analyses of HDACs and HDAC-related deacetylases guide the design of tight-binding inhibitors, and future prospects for developing isozyme-specific inhibitors are quite promising.

Figure 1. Arginase-deacetylase fold

(a) Topology diagrams of arginase, HDAC8, and APAH reveal a common α/β fold with a central, 8-stranded parallel β-sheet (strand order 21387456). The relative positions of metal ligands are indicated on arginase (loops L3, L4, and L7), and HDAC8 and APAH (loops L4 and L7) (each loop is numbered after its preceding β-strand). Green circles indicate residues conserved in arginase, HDAC, APAH, and all related enzymes; yellow circles indicate residues conserved only in arginase and arginase-related metalloenzymes. (b) The Mn²⁺_B site of arginase is conserved in HDAC8, APAH, and related metalloenzymes as D(A,V,L,F)HX_~100D (boldface indicates metal ligands). The Mn²⁺_A site of arginase is not conserved in HDACs or HDAC-related deacetylases. Non-protein metal ligands (red spheres) are solvent molecules in arginase and HDAC8, and the oxygen atoms of a hydroxamate inhibitor in APAH.

Figure 2. Metal binding sites

Structures of HDAC8, HDAC7, and APAH illustrate the binding sites of catalytic metal ion Zn²⁺_A and structural metal ions K⁺_A and K⁺_B (note that the K⁺_B site is occupied by Na⁺ in APAH). Additionally shown are putative inhibitory metal ions Zn²⁺_B in HDAC8 and K⁺_C in APAH, as well as structural metal ion Zn²⁺_B' in HDAC7.

Figure 3. Mode of inhibition by excess K⁺

The "out" conformation of Y323 in the L8 loop of APAH is facilitated by the binding of K⁺_C (magenta sphere) to the adjacent L7 loop. Ligands to K⁺_C include the backbone C=O groups of F286, D289, and S292; the side chain of S292; and two water molecules (red spheres). This third monovalent cation binding site may contribute to the inhibition of enzyme activity at elevated K⁺ concentrations by destabilizing the "in" conformation of Y323 required for catalysis. Although the binding of a third monovalent cation to HDACs has not yet been observed, such a binding mode could similarly account for the inhibitory effects of excess K⁺. Reprinted with permission from reference [24••]. Copyright 2011 American Chemical Society.

Figure 4. Proposed mechanism of HDACs and HDAC-related enzymes

The active site transition metal ion of HDAC8 (Zn²⁺ or Fe²⁺) and general base H143 promote the nucleophilic attack of a metal-bound water molecule at the metal-coordinated C=O group of the acetyl-L-lysine substrate (for clarity, only the side chain of acetyl-L-lysine is shown). As drawn, the nucleophilic lone electron pair on the metal-bound water molecule becomes available only upon proton abstraction, e.g., the electron pair of the breaking O-H bond could add to the π* orbital of the substrate carbonyl, although other molecular orbital explanations are possible. The oxyanion of the tetrahedral intermediate and its flanking transition states are stabilized by metal coordination as well as hydrogen bond interactions with Y306, H143, and H142. H143 serves as a general acid catalyst to facilitate the collapse of the tetrahedral intermediate to form acetate and L-lysine after an intervening proton transfer (possibly mediated by H143). We speculate that the side chain of Y306 may undergo a conformational transition from an "out" conformation to an "in" conformation to accommodate substrate binding and catalysis, based on the conformational mobility of the corresponding residue in related enzymes (Y976 in H976Y HDAC4 [19] and Y323 in APAH [24••]). If so, this could suggest an induced-fit substrate binding mechanism reminiscent of that described for Y248 in carboxypeptidase A [34].

Figure 5. Stereoviews of enzyme-substrate recognition

(a) Molecular recognition of a polypeptide substrate in the active site of Y306F HDAC8 is governed by the side chain of D101, which accepts hydrogen bonds from the backbone NH group of the scissile acetyl-L-lysine residue at position n and the adjacent residue at position n + 1. Residue 101 must be capable of making these dual hydrogen bond interactions for optimal function [30, 31]. Selected active site residues are indicated. Atoms are color-coded as follows: C = green (protein) or yellow (substrate), N = blue, O = red. Metal coordination and hydrogen bond interactions are indicated by black and orange dashed lines, respectively; the catalytic Zn²⁺ ion is a lavender sphere, and the Zn²⁺-bound water molecule is shown as a smaller red sphere. (b) Molecular recognition of a polyamine substrate as observed in the H159A APAH-acetylspermine complex [24••]. The positively-charged amino groups of the substrate make hydrogen bonded salt links with the negatively-charged side chains of E117 and E106 from the adjacent monomer; one secondary amino group also makes an electrostatic interaction with Y168 (3.3 Å, red dashed line) and a cation-π interaction with F225 (green dashed line). The side chain of Y323 may undergo a conformational change to donate a hydrogen bond to the scissile carbonyl linkage. Atoms are color-coded as follows: C = yellow (protein, Y323 "in" conformation), gray (protein, Y323 "out" conformation), olive (substrate), or light brown (E106 from the adjacent monomer in the background, which is not labeled), N = blue, O = red. Metal coordination and hydrogen bond interactions are indicated by black and orange dashed lines, respectively; the catalytic Zn²⁺ ion is a lavender sphere, and water molecules are smaller red spheres.

Figure 6. Zn²⁺ coordination by HDAC inhibitors

(a) Examples of inhibitors capable of forming 4-, 5-, 6-, and 7-membered ring chelates with the catalytic Zn²⁺ ion are shown. It is likely that some of these inhibitors, e.g., the hydroxamic acids, ionize once they are fully bound to the metal ion. (b) Romidepsin and largazole are depsipeptide prodrug HDAC inhibitors that convert into active inhibitory thiols by in vivo reduction and hydrolysis reactions, respectively. The crystal structure of the HDAC8-largazole thiol complex reveals that the thiol side chain of the active inhibitory form of largazole coordinates to the active site Zn²⁺ ion, presumably as the negatively-charged thiolate anion [51•].