Large-scale characterization of post-translational modifications (PTMs), such as phosphorylation, acetylation and ubiquitination, has highlighted their importance in the regulation of a myriad of signaling events. While high-throughput technologies have tremendously helped cataloguing the proteins modified by these PTMs, the identification of lysine-methylated proteins, a PTM involving the transfer of one, two or three methyl groups to the e-amine of a lysine side chain, has lagged behind. While the initial findings were focused on the methylation of histone proteins, several studies have recently identified novel non-histone lysinemethylated proteins. This review provides a compilation of all lysine methylation sites reported to date. We also present key examples showing the impact of lysine methylation and discuss the circuitries wired by this important PTM.

Histone H3 Lys-4 methylation is predominantly catalyzed by a family of methyltransferases whose enzymatic activity depends on their interaction with a three-subunit complex composed of WDR5, RbBP5, and Ash2L. Here, we report that a segment of 50 residues of RbBP5 bridges the Ash2L C-terminal domain to WDR5. The crystal structure of WDR5 in ternary complex with RbBP5 and MLL1 reveals that both proteins binds peptide-binding clefts located on opposite sides of WDR5's β-propeller domain. RbBP5 engages in several hydrogen bonds and van der Waals contacts within a V-shaped cleft formed by the junction of two blades on WDR5. Mutational analyses of both the WDR5 V-shaped cleft and RbBP5 residues reveal that the interactions between RbBP5 and WDR5 are important for the stimulation of MLL1 methyltransferase activity. Overall, this study provides the structural basis underlying the formation of the WDR5-RbBP5 subcomplex and further highlight the crucial role of WDR5 in scaffolding the MLL1 core complex.

The SMYD (SET and MYND domain) family of lysine methyltransferases (KMTs) plays pivotal roles in various cellular processes, including gene expression regulation and DNA damage response. Initially identified as genuine histone methyltransferases, specific members of this family have recently been shown to methylate non-histone proteins such as p53, VEGFR, and the retinoblastoma tumor suppressor (pRb). To gain further functional insights into this family of KMTs, we generated the protein interaction network for three different human SMYD proteins (SMYD2, SMYD3, and SMYD5). Characterization of each SMYD protein network revealed that they associate with both shared and unique sets of proteins. Among those, we found that HSP90 and several of its co-chaperones interact specifically with the tetratrico peptide repeat (TPR)-containing SMYD2 and SMYD3. Moreover, using proteomic and biochemical techniques, we provide evidence that SMYD2 methylates K209 and K615 on HSP90 nucleotide-binding and dimerization domains, respectively. In addition, we found that each methylation site displays unique reactivity in regard to the presence of HSP90 co-chaperones, pH, and demethylation by the lysine amine oxidase LSD1, suggesting that alternative mechanisms control HSP90 methylation by SMYD2. Altogether, this study highlights the ability of SMYD proteins to form unique protein complexes that may underlie their various biological functions and the SMYD2-mediated methylation of the key molecular chaperone HSP90.

Human ASH2L is a trithorax group (TrxG) protein and a regulatory subunit of the SET1 family of lysine methyltransferases. Here we report that Ash2L binds DNA employing a Forkhead-like helix-wing-helix (HWH) domain. In vivo, Ash2L HWH domain is required for binding to the β-globin locus control region (LCR), histone H3 Lys4 tri-methylation and maximal expression of the β-globin gene, validating the functional importance of Ash2L DNA binding activity.

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.