Macrocyclic Octapeptide Binding and Inferences on Protein Substrate Binding to Histone Deacetylase 6

Abstract

Histone deacetylases (HDACs) are essential for the regulation of myriad biological processes, and their aberrant function is implicated in cancer, neurodegeneration, and other diseases. The cytosolic isozyme HDAC6 is unique among the greater family of deacetylases in that it contains two catalytic domains, CD1 and CD2. HDAC6 CD2 is responsible for tubulin deacetylase and tau deacetylase activities, inhibition of which is a key goal as new therapeutic approaches are explored. Of particular interest as HDAC inhibitors are naturally occurring cyclic tetrapeptides such as Trapoxin A or HC Toxin, or the cyclic depsipeptides Largazole and Romidepsin. Even more intriguing are larger, computationally designed macrocyclic peptide inhibitors. Here, we report the 2.0 Å resolution crystal structure of HDAC6 CD2 complexed with macrocyclic octapeptide 1. Comparison with the previously reported structure of the complex with macrocyclic octapeptide 2 reveals that a potent thiolate–zinc interaction made by the unnatural amino acid (S)-2-amino-7-sulfanylheptanoic acid contributes to nanomolar inhibitory potency for each inhibitor. Apart from this zinc-binding residue, octapeptides adopt strikingly different overall conformations and make few direct hydrogen bonds with the protein. Intermolecular interactions are dominated by water-mediated hydrogen bonds; in essence, water molecules appear to cushion the enzyme–octapeptide interface. In view of the broad specificity observed for protein substrates of HDAC6 CD2, we suggest that the binding of macrocyclic octapeptides may mimic certain features of the binding of macromolecular protein substrates.

Chemical Versatility in Catalysis and Inhibition of the Class IIb Histone Deacetylases

“…In addition to illuminating the structural consequences of reactivity for the zinc-bound water molecule, X-ray crystallography has yielded new insight into binding interactions in the outer active site cleft of HDAC6 that contribute to inhibitor affinity and selectivity. For example, the inhibitory potencies and binding modes of cyclic tetrapeptide and depsipeptide natural product inhibitors of HDACs recently inspired the de novo design of macrocyclic octapeptides and nonapeptides exhibiting nanomolar inhibitory potencies. , The zinc-binding group of these inhibitors is the thiol moiety of an unnatural amino acid that displaces the zinc-bound water molecule and coordinates to zinc as the thiolate anion. Similar zinc coordination modes have also been observed for the thiol groups of the marine natural product largazole, the reduced form of the cofactor ( R )-lipoic acid, and a mercaptoacetamide inhibitor. − Intriguingly, the structures of two different macrocyclic peptides complexed with HDAC6 reveal only water-mediated hydrogen bonds between the enzyme and the inhibitor in the outer active site cleft. , The same cushion of water molecules that accommodates the binding of these large inhibitors may similarly accommodate a wide variety of structurally diverse protein substrates.…”

“…For example, the inhibitory potencies and binding modes of cyclic tetrapeptide and depsipeptide natural product inhibitors of HDACs recently inspired the de novo design of macrocyclic octapeptides and nonapeptides exhibiting nanomolar inhibitory potencies. 55,56 The zinc-binding group of these inhibitors is the thiol moiety of an unnatural amino acid that displaces the zinc-bound water molecule and coordinates to zinc as the thiolate anion. Similar zinc coordination modes have also been observed for the thiol groups of the marine natural product largazole, the reduced form of the cofactor (R)-lipoic acid, and a mercaptoacetamide inhibitor.…”

“…57−59 Intriguingly, the structures of two different macrocyclic peptides complexed with HDAC6 reveal only water-mediated hydrogen bonds between the enzyme and the inhibitor in the outer active site cleft. 55,56 The same cushion of water molecules that accommodates the binding of these large inhibitors may similarly accommodate a wide variety of structurally diverse protein substrates.…”