Structure-based prediction of HDAC6 substrates validated by enzymatic assay reveals determinants of promiscuity and detects new potential substrates

Abstract

Histone deacetylases play important biological roles well beyond the deacetylation of histone tails, and therefore have recently been renamed to acetyl-lysine deacetylases (KDACs). In particular, KDAC6 is involved in multiple cellular processes such as apoptosis, cytoskeleton reorganization, and protein folding, affecting substrates such as α-tubulin, Hsp90 and cortactin proteins. We have applied a biochemical enzymatic assay to measure the activity of KDAC6 on a set of candidate unlabeled peptides. These served for the calibration of a structure-based substrate prediction protocol, Rosetta FlexPepBind, previously used for the successful substrate prediction of KDAC8 and other enzymes. The calibration process and comparison of the results between KDAC6 and KDAC8 highlighted structural differences that explain the already reported promiscuity of KDAC6. A proteome-wide screen of reported acetylation sites using our calibrated protocol together with the enzymatic assay provide new peptide substrates and avenues to novel potential functional regulatory roles of this promiscuous, multi-faceted enzyme.

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

“…In addition to protein substrates of HDAC6 CD2 identified through in vivo biochemical methods (Table S5), additional putative protein substrates have been identified by screening peptide fragments in vitro for activity and then correlating peptide sequences with the corresponding proteins for which crystal structures are available. 50,51 Analysis of protein structures identified through this approach yields additional examples of lysine residues contained in loops, α-helices, and one in a β-strand (Table S6).…”