Trimethyllysine (Kme3) reader proteins are targets for inhibition due to their role in mediating gene expression. Although all such reader proteins bind Kme3 in an aromatic cage, the driving force for binding may differ; some readers exhibit evidence for cation–π interactions whereas others do not. We report a general unnatural amino acid mutagenesis approach to quantify the contribution of individual tyrosines to cation binding using the HP1 chromodomain as a model system. We demonstrate that two tyrosines (Y24 and Y48) bind to a Kme3-histone tail peptide via cation–π interactions, but linear free energy trends suggest they do not contribute equally to binding. X-ray structures and computational analysis suggest that the distance and degree of contact between Tyr residues and Kme3 plays an important role in tuning cation–π-mediated Kme3 recognition. Although cation–π interactions have been studied in a number of proteins, this work is the first to utilize direct binding assays, X-ray crystallography, and modeling, to pinpoint factors that influence the magnitude of the individual cation–π interactions.

Lysine crotonylation (Kcr) is a histone post-translational modification that is implicated in numerous epigenetic pathways and diseases. Recognition of Kcr by YEATS domains has been proposed to occur through intermolecular amide−π and alkene−π interactions, but little is known about the driving force of these key interactions. Herein, we probed the recognition of lysine crotonylation and acetylation by the AF9 YEATS domain through incorporation of noncanonical Phe analogs with distinct electrostatics at two positions. We found that amide−π interactions between AF9 and acyllysines are electrostatically tunable, with electron-rich rings providing more favorable interactions. This differs from trends in amide−heteroarene interactions and provides insightful information for therapeutic design. Additionally, we report for the first time that CH−π interactions at Phe28 directly contribute to AF9's recognition of acyllysines, illuminating differences among YEATS domains, as this residue is not highly conserved but has been shown to impart selectivity for specific post-translational modification.

In this work, we experimentally validate that tryptophan provides the strongest cation–π binding interaction among aromatic amino acids and also lend insight into the importance of residue identity in trimethyllysine recognition by reader proteins.