The SET1 family of methyltransferases carries out the bulk of histone H3 Lys-4 methylation in vivo. One of the common features of this family is the regulation of their methyltransferase activity by a tripartite complex composed of WDR5, RbBP5, and Ash2L. To selectively probe the role of the SET1 family of methyltransferases, we have developed a library of histone H3 peptide mimetics and report herein the characterization of an Nα acetylated form of histone H3 peptide (NαH3). Binding and inhibition studies reveal that the addition of an acetyl moiety to the N terminus of histone H3 significantly enhances its binding to WDR5 and prevents the stimulation of MLL1 methyltransferase activity by the WDR5-RbBP5-Ash2L complex. The crystal structure of NαH3 in complex with WDR5 reveals that a high-affinity hydrophobic pocket accommodates the binding of the acetyl moiety. These results provide the structural basis to control WDR5-RbBP5-Ash2L-MLL1 activity and a tool to manipulate stem cell differentiation programs.
SET domain lysine methyltransferases (KMTs) are S‐adenosylmethionine (AdoMet)‐dependent enzymes that catalyze the site‐specific methylation of lysine residues in histones, transcription factors, and other protein substrates. SET domain KMTs also display product specificity, which is defined as their ability to catalyze different degrees of methylation of the lysine‐epsilon amine group, thus imparting an additional hierarchy in methyllysine signaling. To understand the mechanism underlying product specificity, we have characterized two active site mutants of the monomethylase SET7/9 that alter its specificity to a dimethylase and a trimethylase, respectively. Structures of the SET7/9 mutants in complex with peptides bearing unmodified, mono‐, di‐, and trimethylated lysines reveal that water molecules within the active site function as place holders that linearly align the lysine epsilon‐amine group with the methyl group of AdoMet to promote methyltransfer. As the methylation state of the lysine substrate increases during successive reactions, the water molecules dissociate from the active site, thereby enlarging the lysine binding channel to accommodate the increasing bulk of the methylated epsilon‐amine group. Collectively, our findings illuminate the catalytic roles of active site water molecules in facilitating lysine multiple methylation by SET domain KMTs.
