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N ε -Methylation of lysine residues in histones plays an essential role in the regulation of eukaryotic transcription. The 'highest' methylation mark, N ε -trimethyllysine, is specifically recognised by N ε -trimethyllysine binding 'reader' domains, and undergoes demethylation, as catalysed by 2-oxoglutarate dependent JmjC oxygenases. We report studies on the recognition of the closest positively charged N ε -trimethyllysine analogue, i.e. its trimethylphosphonium derivative (K P me 3 ), by N ε -trimethyllysine histone binding proteins and N ε - trimethyllysine demethylases. Calorimetric and computational studies with histone binding proteins reveal that H3K P 4me 3 binds more tightly than the natural H3K4me 3 substrate, though the relative differences in binding affinity vary. Studies with JmjC demethylases show that some, but not all, of them can accept the phosphonium analogue of their natural substrates and that the methylation state selectivity can be changed by substitution of nitrogen for phosphorus. The combined results reveal that very subtle changes, e.g . substitution of nitrogen for phosphorus, can substantially affect interactions between ligand and reader domains / demethylases, knowledge that we hope will inspire the development of highly selective small molecules modulating their activity., Competing Interests: Competing interests The authors declare no competing interests.

The understanding of biomolecular recognition of posttranslationally modified histone proteins is centrally important to the histone code hypothesis. Despite extensive binding and structural studies on the readout of histones, the molecular language by which posttranslational modifications on histone proteins are read remains poorly understood. Here we report physical-organic chemistry studies on the recognition of the positively charged trimethyllysine by the electron-rich aromatic cage containing PHD3 finger of KDM5A. The aromatic character of two tryptophan residues that solely constitute the aromatic cage of KDM5A was fine-tuned by the incorporation of fluorine substituents. Our thermodynamic analyses reveal that the wild-type and fluorinated KDM5A PHD3 fingers associate equally well with trimethyllysine. This work demonstrates that the biomolecular recognition of trimethyllysine by fluorinated aromatic cages is associated with weaker cation-π interactions that are compensated by the energetically more favourable trimethyllysine-mediated release of high-energy water molecules that occupy the aromatic cage., (© 2020. The Author(s).)