This feature article highlights the application of chemical tools for understanding and manipulating the histidine residues in proteins.

Histone lysine methacrylation and crotonylation are epigenetic marks that play important roles in human gene regulation. Here, we explore the molecular recognition of histone H3 peptides possessing methacryllysine and crotonyllysine at positions 18 and 9 (H3K18 and H3K9) by the AF9 YEATS domain. Our binding studies demonstrate that the AF9 YEATS domain displays a higher binding affinity for histones possessing crotonyllysine than the isomeric methacryllysine, indicating that AF9 YEATS distinguishes between the two regioisomers. Molecular dynamics simulations reveal that the crotonyllysine/methacryllysine-mediated desolvation of the AF9 YEATS domain provides an important contribution to the recognition of both epigenetic marks. These results provide important knowledge for the development of AF9 YEATS inhibitors, an area of biomedical interest.

SETD3-catalysed N³-methylation of His73 in β-actin plays a key role in stabilisation of actin filaments in the metazoan cells. Overexpression and/or dysregulation of SETD3 is associated with several human pathologies, including cancer. Here, we examined the role of the Ile71 residue in β-actin on human SETD3 catalysis. Substitution of Ile71 in β-actin peptides by its natural and unnatural mimics reveals that the ‘secondary’ Ile71 binding pocket modulates the substrate efficiency of β-actin. Our enzymatic work demonstrates that human SETD3 can accommodate structurally diverse hydrophobic side chains in its Ile71 binding pocket, providing clear limits of the size and shape of Ile analogues. Water thermodynamics calculations reveal that the Ile71 pocket is occupied by high-energy water molecules, that are released upon the Ile71 binding, contributing favourably to the SETD3-βA complex formation. The work highlights that the hydrophobic Ile71 binding site plays an essential role in SETD3 catalysis, contributing to an ongoing effort in the design and development of chemical probes targeting SETD3.

Lysine acylations on histones and their recognition by chromatin-binding reader domains and removal by histone deacylases function as an important mechanism for eukaryotic gene regulation. Histone lysine crotonylation (Kcr) is an epigenetic mark associated with active transcription, and its installation and removal are dynamically regulated by cellular epigenetic enzymes. Here, we report binding studies and enzyme assays with histone H3K9 peptides bearing simplest Kcr analogs with varying hydrocarbon chain length, bulkiness, rigidity and polarity. We demonstrate that the AF9 YEATS domain displays selectivity for binding of different acylation modifications on histone H3K9 peptides and exhibits preference for bulkier cinnamoylated lysine over crotonylated lysine and its mimics. SIRT2 shows deacylase activity against most of acylated H3K9 peptides bearing different crotonyllysine mimics, however, it displays a poor ability for the removal of cinnamoyl and trifluorocrotonyl groups. These results demonstrate different substrate selectivities of epigenetic proteins acting on crotonyllysine and pave the way for rational design and development of AF9 YEATS and SIRT2 inhibitors for treatment of human diseases, including cancer.

N^τ‐methylation of His73 in actin by histidine methyltransferase SETD3 plays an important role in stabilising actin filaments in eukaryotes. Mutations in actin and overexpression of SETD3 have been related to human diseases, including cancer. Here, we investigated the importance of Trp79 in β‐actin on productive human SETD3 catalysis. Substitution of Trp79 in β‐actin peptides by its chemically diverse analogues reveals that the hydrophobic Trp79 binding pocket modulates the catalytic activity of SETD3, and that retaining a bulky and hydrophobic amino acid at position 79 is important for efficient His73 methylation by SETD3. Molecular dynamics simulations show that the Trp79 binding pocket of SETD3 is ideally shaped to accommodate large and hydrophobic Trp79, contributing to the favourable release of water molecules upon binding. Our results demonstrate that the distant Trp79 binding site plays an important role in efficient SETD3 catalysis, contributing to the identification of new SETD3 substrates and the development of chemical probes targeting the biomedically important SETD3.