Discovery of Substrates for a SET Domain Lysine Methyltransferase Predicted by Multistate Computational Protein Design

• Published in: Structure (London) 2015-01, Vol.23 (1), p.206-215
• Main Authors: Lanouette, Sylvain, Davey, James A., Elisma, Fred, Ning, Zhibin, Figeys, Daniel, Chica, Roberto A., Couture, Jean-François
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Computational Biology - methods; HEK293 Cells; Histone-Lysine N-Methyltransferase - chemistry; Histone-Lysine N-Methyltransferase - genetics; Histone-Lysine N-Methyltransferase - metabolism; Humans; Models, Molecular; Mutagenesis, Site-Directed; Protein Engineering - methods; Protein Interaction Domains and Motifs - genetics; Protein Interaction Maps; Protein Structure, Tertiary - genetics; Substrate Specificity
• ISSN: 0969-2126; 1878-4186
• DOI: 10.1016/j.str.2014.11.004

Characterization of lysine methylation has proven challenging despite its importance in biological processes such as gene transcription, protein turnover, and cytoskeletal organization. In contrast to other key posttranslational modifications, current proteomics techniques have thus far shown limited success at characterizing methyl-lysine residues across the cellular landscape. To complement current biochemical characterization methods, we developed a multistate computational protein design procedure to probe the substrate specificity of the protein lysine methyltransferase SMYD2. Modeling of substrate-bound SMYD2 identified residues important for substrate recognition and predicted amino acids necessary for methylation. Peptide- and protein- based substrate libraries confirmed that SMYD2 activity is dictated by the motif [LFM]−1-K∗-[AFYMSHRK]+1-[LYK]+2 around the target lysine K∗. Comprehensive motif-based searches and mutational analysis further established four additional substrates of SMYD2. Our methodology paves the way to systematically predict and validate posttranslational modification sites while simultaneously pairing them with their associated enzymes. [Display omitted] •Multistate design predicts substrates for posttranslational modifying enzymes•Multistate design recapitulates the motif determined by semidegenerate peptide arrays•SMYD2 methylates recognizes the motif [LFM]−1-K∗-[AFYMSHRK]+1-[LYK]+2•SMYD2 methylates transcription factors and chromatin remodeling complexes in vitro Characterization of lysine methylation has proven challenging despite its biological importance. Lanouette et al. use multistate design (MSD) to predict substrates for a protein lysine methyltransferase, and show that MSD is a facile method for determination of target sequences for posttranslational modifying enzymes.