Substrate binding to histone deacetylases as shown by the crystal structure of the HDAC8–substrate complex

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Abstract
Histone deacetylases (HDACs)—an enzyme family that deacetylates histones and non‐histone proteins—are implicated in human diseases such as cancer, and the first‐generation of HDAC inhibitors are now in clinical trials. Here, we report the 2.0 Å resolution crystal structure of a catalytically inactive HDAC8 active‐site mutant, Tyr306Phe, bound to an acetylated peptidic substrate. The structure clarifies the role of active‐site residues in the deacetylation reaction and substrate recognition. Notably, the structure shows the unexpected role of a conserved residue at the active‐site rim, Asp 101, in positioning the substrate by directly interacting with the peptidic backbone and imposing a constrained cis ‐conformation. A similar interaction is observed in a new hydroxamate inhibitor–HDAC8 structure that we also solved. The crucial role of Asp 101 in substrate and inhibitor recognition was confirmed by activity and binding assays of wild‐type HDAC8 and Asp101Ala, Tyr306Phe and Asp101Ala/Tyr306Phe mutants.