Trimethyllysine reader proteins exhibit widespread charge-agnostic binding via different mechanisms to cationic and neutral ligands

In the last 40 years, cation−π interactions have become part of the lexicon of noncovalent forces that drive protein binding. Indeed, tetraalkylammoniums are universally bound by aromatic cages in proteins, suggesting that cation−π interactions are a privileged mechanism for binding these ligands. A prominent example is the recognition of histone trimethyllysine (Kme3) by the conserved aromatic cage of reader proteins, dictating gene expression. However, two proteins have recently been sug-gested as possible exceptions to conventional understanding of tetraalkylammonium recognition. To broadly interrogate the role of cation−π interactions in protein binding interactions, we report the first large-scale comparative evaluation of reader proteins for a neutral Kme3 isostere, experimental and computational mechanistic studies, and structural analysis. We find unexpected widespread binding of readers to a neutral isostere, with no single factor dictating charge selectivity, demonstrat-ing the challenge to predict such interactions. Further, readers that bind both cationic and neutral ligands display an unprece-dented change in mechanism: binding Kme3 via cation−π interactions and the neutral isostere through the hydrophobic effect in the same aromatic cage. This discovery challenges traditional understanding of molecular recognition of tetraalkylammo-niums by aromatic cages in myriad protein-ligand interactions and establishes a new framework for selective inhibitor design by exploiting differences in charge-dependence.