Lysine methylation of histone proteins is a common and well-recognized means of epigenetic regulation. The proteins responsible for binding and interpreting these post-translation modifications (PTMs) are called “reader” proteins. PTMs and the associated reader proteins are essential for regulation of gene transcription and its downstream effects including proper development and cell differentiation as well as the development of disease states. Reader proteins that recognize methylation states of lysine do so using a cage of aromatic residues to bind methyllysine residues via cation-pi interactions.<br/><br/>Here, we used genetic code expansion and peptidic inhibitors to probe the interactions involved in trimethyllysine binding in the chromodomain of human protein chromobox protein 5 (CBX5). CBX5 binds trimethyllysine at position 9 of histone 3 utilizing an aromatic cage made of one tyrosine (Y4), one phenylalanine (F28), and one tryptophan. Using genetic code expansion, we were able to site-specifically install non-canonical amino acids with different electronic properties within the aromatic cage in order to elucidate the impact of cation-pi interactions and the individual contributions of both Y4 and F28 positions to binding. Using peptidic inhibitors with varying substituents at the methyllysine position, we were able to evaluate the effects of different methyllysine mimetics on CBX5 binding. Binding was monitored by isothermal titration calorimetry with the ability to correlate properties of each non-canonical amino acid and methyllysine mimetic with binding energy.<br/><br/>These systematic studies allow us to identify discrete contributions to binding from both ligand and protein. This not only contributes to our fundamental understanding of reader proteins but will also inform the design of further inhibitors to target these important proteins for human health.

Probing interactions in methyllysine reader proteins using genetic code expansion

2019 Fall National Meeting

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