X-ray Crystallographic Snapshots of Substrate Binding in the Active Site of Histone Deacetylase 10

Abstract

Histone deacetylase 10 (HDAC10) is a zincdependent polyamine deacetylase enriched in the cytosol of eukaryotic cells. The active site of HDAC10 contains catalytic residues conserved in other HDAC isozymes that function as lysine deacetylases: Y307 assists the zinc ion in polarizing the substrate carbonyl for nucleophilic attack, and the H136-H137 dyad serves general base−general acid functions. As an inducer of autophagy, HDAC10 is an attractive target for the design of selective inhibitors that may be useful in cancer chemotherapy. Because detailed structural information regarding the catalytic mechanism of HDAC10 may inform new approaches to inhibitor design, we now report X-ray crystal structures of HDAC10 in which reaction intermediates with substrates N 8 -acetylspermidine and N-acetylputrescine are trapped in the active site. The Y307F substitution prevents activation of the substrate carbonyl for nucleophilic attack by the zinc-bound water molecule, thereby enabling crystallographic isolation of intact enzyme−substrate complexes. The H137A substitution removes the catalytically obligatory general acid, thereby enabling crystallographic isolation of oxyanionic tetrahedral intermediates. Finally, the acetate complex with the wild-type enzyme represents a product complex after dissociation of the polyamine coproduct. Taken together, these structures provide snapshots of the reaction coordinate of acetylpolyamine hydrolysis and are consistent with a mechanism in which tandem histidine residues H136 and H137 serve as general base and general acid catalysts, respectively. The function of the histidine dyad in the HDAC10 mechanism appears to be similar to that in HDAC6, but not HDAC8 in which both functions are served by the second histidine of the tandem pair.

Aza-SAHA Derivatives are Selective Histone Deacetylase 10 Chemical Probes That Inhibit Polyamine Deacetylation

“…The Zn 2+ ligand H176 donates a hydrogen bond to E274 as well, as also observed in the HDAC10-tubastatin A complex (11), thereby forming a hydrogen bond network between H176 and the anilide NH group. Water-mediated hydrogen bond networks with H176 are also observed in other HDAC10-inhibitor complexes (11,37). The 2.15 Å-resolution crystal structure of the HDAC10-14 complex (Figure 4B) reveals bidentate hydroxamate-Zn 2+ coordination with C=O---Zn 2+ and N-O ----Zn 2+ separations of 2.1 Å and 2.3 Å, respectively.…”

“…Possibly, water #194 contributes to increased affinity. Structures of inactivated HDAC10 complexed with substrates reveal that 1-2 water molecules engage the secondary ammonium group of N 8 -acetylspermidine and the primary ammonium group of N-acetylputrescine in hydrogen bond interactions that dictate substrate specificity (37). The implicated protons on these ammonium cations point directly to water molecules that are also stabilized by hydrogen bonds with E274, much like the proton on the tertiary ammonium cation of DKFZ-711 that donates a hydrogen bond to water #194.…”

Identification of HDAC10 Inhibitors that Modulate Autophagy-Related Proteins in Transformed Cells

“…The selection of capping groups as well as the position of the basic amine was guided by docking the compounds into the available X-ray structures of danio rerio HDAC10 (drHDAC10). 18,22,23 Introducing a methylene-group between the benzhydroxamic core and the basic amine resulted in hits that showed a salt-bridge to the gatekeeper residue Glu274 of HDAC10 (Fig. S1, Supplementary Information) and were therefore prepared as described in the Methods section.…”

First fluorescent acetylspermidine deacetylation assay for HDAC10 identifies selective inhibitors with cellular target engagement

“…The crystal structure of inactivated drHDAC10 complexed with N 8 -acetylspermidine shows that Glu274 engages the protonated secondary amino group of the substrate with two water-mediated hydrogen bonds. 14 The preferential binding of N 8 -acetylspermidine versus N 1 -acetylspermidine is explained by the position and orientation of the secondary amino group. A distance of four carbons between the amide moiety and the secondary amino group is favorable for the substrate recognition.…”