Structural and Functional Analysis of the Human HDAC4 Catalytic Domain Reveals a Regulatory Structural Zinc-binding Domain

Abstract

Histone deacetylases (HDACs) regulate chromatin status and gene expression, and their inhibition is of significant therapeutic interest. To date, no biological substrate for class IIa HDACs has been identified, and only low activity on acetylated lysines has been demonstrated. Here, we describe inhibitor-bound and inhibitor-free structures of the histone deacetylase-4 catalytic domain (HDAC4cd) and of an HDAC4cd active site mutant with enhanced enzymatic activity toward acetylated lysines. The structures presented, coupled with activity data, provide the molecular basis for the intrinsically low enzymatic activity of class IIa HDACs toward acetylated lysines and reveal active site features that may guide the design of class-specific inhibitors. In addition, these structures reveal a conformationally flexible structural zinc-binding domain conserved in all class IIa enzymes. Importantly, either the mutation of residues coordinating the structural zinc ion or the binding of a class IIa selective inhibitor prevented the association of HDAC4 with the N-CoR⅐HDAC3 repressor complex. Together, these data suggest a key role of the structural zinc-binding domain in the regulation of class IIa HDAC functions.

Plants Release Precursors of Histone Deacetylase Inhibitors to Suppress Growth of Competitors

“…We first assessed in silico the binding potential of hydroxamic acids to different HDAC proteins, based on the high sequence conservation of HDACs across organisms (Supplemental Figure 2). In comparative analyses of docking into the crystal structures of human class I and class II HDACs and of bacterial histone deacetylase-like protein (HDLP) (Finnin et al, 1999;Somoza et al, 2004;Bottomley et al, 2008), APO and AMPO behaved similarly to TSA and SAHA (Supplemental Figure 3). In the absence of available crystal structures for plant HDACs, we performed docking analyses for these enzymes using homology models of Arabidopsis HDA6 (class I) and HDA2 (class III) (Supplemental Figure 4).…”

“…Sequences of human HDACs were obtained from PDB IDs 1T64 (Somoza et al, 2004) and 2VQM (Bottomley et al, 2008); the HDLP sequence of A. aeolicus was obtained from PDB ID 1C3R (Finnin et al, 1999); Arabidopsis sequences were retrieved from The Arabidopsis Information Resource (Rhee et al, 2003) using gene models AT4G38130.1 (HDA1/19, class I), AT5G63110.1 (HDA6, class I), and AT5G26040.2 (HDA2, class III).…”

“…Conformations of the ligands suitable for docking were obtained by energy minimization using the MMFF94 force field (Halgren, 1996) as implemented in BALLView (Moll et al, 2006). In the case of HDAC4, two water molecules have been reported to mediate the binding of a hydroxamic acid inhibitor (Bottomley et al, 2008). Both water molecules (A 2296 and A 2375 in PDB ID 2VQM) were retained in the binding site, and their respective hydrogen positions were optimized with the MMFF94 force field in context of the binding site with the bound hydroxamic acid inhibitor.…”

“…For HDAC8, the binding site of PDB ID 1T64 (Somoza et al, 2004), a human HDAC8 structure with bound inhibitor TSA, was used and preprocessed (side chain orientation, protonation state) for docking with Reduce version 3.10 (Word et al, 1999). For human HDAC4, PDB-ID 2VQM (Bottomley et al, 2008) was used, a structure with a bound hydroxamate inhibitor. For the A. aeolicus HDLP, PDB ID 1C3R (Finnin et al, 1999) with bound TSA was used.…”

Vorinostat-Like Molecules as Structural, Stereochemical, and Pharmacological Tools

“…[5][6][7] To date, detailed biostructural information is available only regarding a few among the known 11 metallo-enzymes. 1,[8][9][10][11] Consequently, the design of isoform-and class-selective inhibitors is somewhat arbitrary and derivative. 1,7,[12][13][14] In addition, complete data regarding the factual HDAC profile for most of the early discovered agents are not available, since the development of effective isozyme-based assays is relatively recent.…”