Development of Lysine Crotonyl‐Mimic Probe to Covalently Identify H3K27Cr Interacting Proteins

Histone lysine crotonylation (Kcr) is one newly discovered acylation modification and regulates numerous pathophysiological processes. The binding affinity between Kcr and its interacting proteins is generally weak, which makes it difficult to effectively identify Kcr‐interacting partners. Changing the amide of crotonyl to an ester increased reactivity with proximal cysteines and retained specificity for Kcr antibody. The probe “H3g27Cr” was designed by incorporating the ester functionality into a H3K27 peptide. Using this probe, multiple Kcr‐interacting partners including STAT3 were successfully identified, and this has not been reported previously. Further experiments suggested that STAT3 possibly could form complexes with Histone deacetylase HDACs to downregulate the acetylation and crotonylation of Histone H3K27. Our unique design provided intriguing tools to further explore Kcr‐interacting proteins and elucidate their working mechanisms. The enhancement of cysteine reactivity of crotonylated lysine (Kcr) in thiol‐Michael addition by replacing the amide bond with an ester bond is reported. The H3g27Cr probe identified the known H3K27Cr‐binding protein HDAC1, as well as the unknown protein STAT3, as potential binders of H3K27Cr in a possibly complex manner. A specific crotonyl group aimed at identifying newer Kcr‐binding interactomes is reported.