Histone lysine methylation plays a critical role in the organization and regulation of the eukaryotic genome via association with reader proteins that sense the post-translational modification and induce recruitment of additional protein complexes. Dysregulation of lysine methylation has been implicated in a range of diseases, therefore understanding the mechanism of recognition is of interest for therapeutic development. Readers that recognize methylated lysine contain a conserved aromatic cage motif through which binding is driven by cation-π interactions. Interestingly, PHD domains that selectively recognize trimethyllysine uniquely contain a conserved methionine residue that occupies the back face of the aromatic cage. While sulfur-arene interactions have grown in appreciation as prominent noncovalent interactions in proteins, the existence of sulfur-cation interactions has yet to be explored. Our goal is to probe this interaction via simultaneous investigations using peptide and protein systems. By incorporating methionine analogs that vary in electrostatic potential, polarizability, and hydrophobicity, we are able to directly tune the influence these parameters have on the interaction with the trimethyllysine cation. In a β-hairpin system, methionine and its analogs are placed cross-strand from trimethyllysine. Through a series of double mutant cycles, the magnitude of the sidechain-sidechain interaction is measured by NMR. We are also studying this interaction in two PHD domains, TAF3 and DIDO1. Harnessing the promiscuity of the methionine tRNA synthetase, we have incorporated norleucine and selenomethionine, and mutated methionine to alanine and cysteine within the aromatic cage. Changes in binding affinity are then measured by isothermal titration calorimetry. In both systems, there is a positive linear free energy relationship between the polarizability of the methionine analog and Gibbs free energy for this interaction, where increased polarizability of the analog correlates with more favorable interactions with trimethyllysine. Characterizing the role of sulfur-cation interactions will help us better comprehend their implications for downstream signaling with other epigenetic protein complexes and add new insight into the function of reader domains, as well as uncover a new noncovalent interaction that could be involved in protein folding and other protein-protein interactions more broadly.

Role of methionine in recognition of trimethyllysine by epigenetic reader proteins

Macromolecular Chemistry: The Second Century

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ACS Spring 2020 National Meeting & Expo

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.