Unnatural Amino Acid Chemistry for Lysine Methyltransferase Substrate Discovery

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Project Abstract Protein lysine methylation represents a prominent post-translational modification in biology. This modification occurs in a multitude of proteins, including histones, transcription factors, chromatin modifying enzymes, ribosomal proteins, cytoplasmic signaling enzymes, chaperones, spliceosomal factors, and cytoskeletal proteins. Lysine methylation frequently modulates protein:protein interactions, often through the recruitment of methyllysine binding factors, and has been implicated in regulating a diverse array of biological phenomena, such as transcription, translation, DNA damage response, signal transduction, and protein chaperone function. These modifications are catalyzed by S-adenosylmethionine (AdoMet)-dependent lysine methyltransferases (KMTs), the majority of which belong to the SET domain family. The human genome encodes over 50 predicted SET domain KMTs. It is fundamentally important to elucidate the substrate selectivities of these enzymes, as methylation of their substrates defines their respective biological functions. Toward this goal, several techniques have been developed to facilitate substrate identification of KMTs, including candidate-based approaches, peptide and protein arrays, and a chemical affinity-mass spectrometry technique that utilizes AdoMet analogs derivatized with bio-orthogonally reactive groups. Although these methods have aided in characterizing the substrate selectivities of certain KMTs, substrate identification remains a persistent challenge. The Gozani (Stanford University), Trievel (University of Michigan), Mehl (Oregon State University), and Larsen (University of Michigan) laboratories have established an interdisciplinary collaboration to devise and implement a novel method for discovering protein substrates of SET domain KMTs. This new approach is complementary to current techniques and is based upon the introduction of an electrophilic unnatural amino acid (UAA) in the active sites of KMTs. This UAA will facilitate proximity-induced chemical crosslinking with the lysine epsilon amino group in protein substrates, with subsequent identification of the crosslinked substrates by mass spectrometry. We envision that this methodology will enable systematic characterization of the substrate selectivities of SET domain KMTs, yielding key insights into their biological functions and how dysregulation of these enzymes may contribute to aberrant protein methylation and disease.