SET7/9 Catalytic Mutants Reveal the Role of Active Site Water Molecules in Lysine Multiple Methylation

Abstract

SET domain lysine methyltransferases (KMTs) methylate specific lysine residues in histone and non-histone substrates. These enzymes also display product specificity by catalyzing distinct degrees of methylation of the lysine ⑀-amino group. To elucidate the molecular mechanism underlying this specificity, we have characterized the Y245A and Y305F mutants of the human KMT SET7/9 (also known as KMT7) that alter its product specificity from a monomethyltransferase to a di-and a trimethyltransferase, respectively. Crystal structures of these mutants in complex with peptides bearing unmodified, mono-, di-, and trimethylated lysines illustrate the roles of active site water molecules in aligning the lysine ⑀-amino group for methyl transfer with S-adenosylmethionine. Displacement or dissociation of these solvent molecules enlarges the diameter of the active site, accommodating the increasing size of the methylated ⑀-amino group during successive methyl transfer reactions. Together, these results furnish new insights into the roles of active site water molecules in modulating lysine multiple methylation by SET domain KMTs and provide the first molecular snapshots of the mono-, di-, and trimethyl transfer reactions catalyzed by these enzymes.SET domain enzymes represent a family of S-adenosylmethionine (AdoMet) 3 -dependent methyltransferases that catalyze the site-specific methylation of protein lysyl residues in a host of proteins, including histones, transcription factors, chromatin-modifying enzymes, ribosomal subunits, and other substrates (1-3). In many instances, these modifications serve to recruit effector proteins that recognize methyl-lysyl residues in a sequence-dependent fashion (4). In addition, SET domain KMTs exhibit product specificity, defined as their ability to catalyze mono-, di-, or trimethylation of the lysine ⑀-amino group. This specificity is biologically relevant because many methyllysine-binding proteins can discriminate among different degrees of lysine methylation (4). Thus, both the site and degree of lysine methylation are critical to recognition by effector proteins.Structural and functional studies have identified a Phe/Tyr switch in the active site of SET domain KMTs that governs their respective product specificities (5, 6). According to this model, KMTs that possess a tyrosine in the Phe/Tyr switch site are limited to catalyzing lysine monomethylation, whereas enzymes that possess a phenylalanine or another hydrophobic residue in this position display di-or trimethyltransferase activity. Mutational analysis of various SET domain KMTs, including DIM-5 (KMT1) (6), SET7/9 (6), G9A (KMT1C) (5), and SET8 (also known as PR-SET7 and KMT5A) (7,8), has demonstrated that substitutions in the Phe/Tyr switch result in predictable changes in product specificity. Several models have been proposed to explain the mechanism by which the Phe/Tyr switch site governs this specificity, including variations in the diameter of the active site due to the size of Phe/Tyr switch residue and steric hindrance by t...

Mutation of A677 in histone methyltransferase EZH2 in human B-cell lymphoma promotes hypertrimethylation of histone H3 on lysine 27 (H3K27)

“…3 and Table S2). Similar to other methyltransferases, such as Set7/9, the hydroxyl group of the highly conserved EZH2 Y641 residue appears to optimally orient the unmethylated and monomethylated forms of the H3K27 substrate for methyl transfer through a hydrogen bond with the ε-amine group of H3K27 (34,35). However, with ∼3.3 Å between the hydroxyl oxygen of Y641 and the ε-amine group of H3K27me2, the model shows very little room for the dimethylated lysine to rotate into position to accept a third methyl group.…”

A687V EZH2 Is a Driver of Histone H3 Lysine 27 (H3K27) Hypertrimethylation

“…Although there has not been much work focused on EZH2 A687, or the equivalent residue in other enzymes, this proposal is supported by extensive work with SET domain F/Y switch residues that also coordinate the active site water molecule. In SET7/9, this residue is a tyrosine (Y305) and mutation to a phenylalanine increases the amount of Kme2 product (34). Conversely, in G9A, this residue is a phenylalanine (F1152) and mutation to a tyrosine reduces the amount of Kme2 product (36).…”

“…This is accomplished in large part by the highly conserved tyrosine residue (Y641 in EZH2, Y245 in SET7/9; Supplementary Fig. S7) and the active site water molecule as evidenced by the loss of activity with unmethylated lysine substrates in the EZH2 Y641N/F/S/H/C and SET7/9 Y245A mutants (19,21,22,34). For the me1!me2 reaction, the lysine substrate must be oriented with the Kme1 methyl group rotated away from the methyl transfer pore so that a second methyl group may be transferred (Fig.…”

“…This step is limited by steric hindrance between the large Kme2 substrate and EZH2 Y641, SET7/9 Y245, or G9a Y1067. Mutation of this restrictive tyrosine to a smaller residue converts these enzymes into efficient trimethyltransferases (19,21,22,34,35).…”

Structure-guided mutational analysis reveals the functional requirements for product specificity of DOT1 enzymes

“…In contrast, combinations of structure studies and mutational analysis of SET KMTs have been very successful in determining the molecular mechanism of their catalytic activity [22][23][24][25][26][27][28] . We used Trypanosoma brucei, a unicellular parasitic eukaryote that causes African sleeping sickness, as a model system to shed light on the methylation state-specific action of DOT1 enzymes.…”