Protein lysine methylation is a distinct class of post-translational modifications because it minimally alters the size and positive charge of the lysine side chain. In cellular contexts, the human genome encodes over 60 protein lysine methyltransferases (PKMTs), with the S-adenosyl-l-methionine (SAM) cofactor as the methyl donor, to modify thousands of lysine sites on histones and nonhistone targets in a highly orchestrated manner. The biological roles of protein lysine methylation are increasingly implicated in epigenetic regulation to define diverse cell fates, and their dysregulation is frequently associated with developmental abnormalities and various aspects of cancerous malignancy. However, it has been challenging to annotate the multiple upstream methyltransferase(s) in parallel from known methyllysine marks in the context of over 60 PKMT candidates with redundant and cell-type-dependent activities. We therefore envisioned the technology of Covalent Trapping of Protein Lysine Methyltransferases (CTPM) by assembling the ternary dead-end complex of PKMTs with substrate-cofactor surrogates. With SET-domain-containing PKMTs, the norleucine(Nle)-SAM pair was shown to be a robust structural motif to form such dead-end complexes, likely via harnessing the common feature of the transition state of PKMT-catalyzed lysine methylation. Our CTPM peptidic probes contain the Nle warhead in the place of substrate lysine, the photo-cross-linking residue in proximity of Nle, and the terminal biotin anchor for target enrichment. These CTPM probes, upon pairing with the SAM cofactor, show high efficiency in trapping the upstream PKMTs of the cognate histone and nonhistone substrates.
Zong et al. (Wed,) studied this question.