Catalytic roles for carbon-oxygen hydrogen bonding in SET domain lysine methyltransferases

SET domain enzymes represent a distinct family of protein lysine methyltransferases in eukaryotes. Recent studies have yielded significant insights into the structural basis of substrate recognition and the product specificities of these enzymes. However, the mechanism by which SET domain methyltransferases catalyze the transfer of the methyl group from S-adenosyl-L-methionine to the lysine epsilon-amine has remained unresolved. To elucidate this mechanism, we have determined the structures of the plant SET domain enzyme, pea ribulose-1,5 bisphosphate carboxylase/oxygenase large subunit methyltransferase, bound to S-adenosyl-L-methionine, and its non-reactive analogs Aza-adenosyl-L-methionine and Sinefungin, and characterized the binding of these ligands to a homolog of the enzyme. The structural and biochemical data collectively reveal that S-adenosyl-L-methionine is selectively recognized through carbon-oxygen hydrogen bonds between the cofactor's methyl group and an array of structurally conserved oxygens that comprise the methyl transfer pore in the active site. Furthermore, the structure of the enzyme co-crystallized with the product epsilon-N-trimethyllysine reveals a trigonal array of carbon-oxygen interactions between the epsilon-ammonium methyl groups and the oxygens in the pore. Taken together, these results establish a central role for carbon-oxygen hydrogen bonding in aligning the cofactor's methyl group for transfer to the lysine epsilon-amine and in coordinating the methyl groups after transfer to facilitate multiple rounds of lysine methylation.     

Three-dimensional structures of fibrillar Sm proteins: Hfq and other Sm-like proteins

SET domain lysine methyltransferases are known to catalyze site and state-specific methylation of lysine residues in histones that is fundamental in epigenetic regulation of gene activation and silencing in eukaryotic organisms. Here we report the three-dimensional solution structure of the SET domain histone lysine methyltransferase (vSET) from Paramecium bursaria chlorella virus 1 bound to cofactor S-adenosyl-L-homocysteine and a histone H3 peptide containing mono-methylated lysine 27. The dimeric structure, mimicking an enzyme/cofactor/substrate complex, yields the structural basis of the substrate specificity and methylation multiplicity of the enzyme. Our results from mutagenesis and enzyme kinetics analyses argue that a general base mechanism is less likely for lysine methylation by SET domains; and that the only invariant active site residue tyrosine 105 in vSET facilitates methyl transfer from cofactor to the substrate lysine by aligning intermolecular interactions in the lysine access channel of the enzyme.     

Mechanism of histone lysine methyl transfer revealed by the structure of SET7/9-AdoMet

The methylation of lysine residues of histones plays a pivotal role in the regulation of chromatin structure and gene expression. Here, we report two crystal structures of SET7/9, a histone methyltransferase (HMTase) that transfers methyl groups to Lys4 of histone H3, in complex with S-adenosyl-L-methionine (AdoMet) determined at 1.7 and 2.3 A resolution. The structures reveal an active site consisting of: (i) a binding pocket between the SET domain and a c-SET helix where an AdoMet molecule in an unusual conformation binds; (ii) a narrow substrate-specific channel that only unmethylated lysine residues can access; and (iii) a catalytic tyrosine residue. The methyl group of AdoMet is directed to the narrow channel where a substrate lysine enters from the opposite side. We demonstrate that SET7/9 can transfer two but not three methyl groups to unmodified Lys4 of H3 without substrate dissociation. The unusual features of the SET domain-containing HMTase discriminate between the un- and methylated lysine substrate, and the methylation sites for the histone H3 tail.     

SET8 recognizes the sequence RHRK20VLRDN within the N terminus of histone H4 and mono-methylates lysine 20

Methylation of lysine 20 in histone H4 has been proven to play important roles in chromatin structure and gene regulation. SET8 is one of the methyltransferases identified to be specific for this modification. In this study, the minimal active SET domain of SET8 has been mapped to the region of amino acids 195-352. This region completely retains the same methylation activity and substrate specificity as the full-length SET8. The SET domain recognizes a stretch of specific amino acid sequence around lysine 20 of H4 for its methylation activity. Methylation assays with N terminus mutants of H4 that contain deletions and single alanine or glutamine substitutions of charged residues revealed that SET8 requires the sequence RHRK20VLRDN for methylation at lysine 20. The individual mutation of any charged residue in this sequence to alanine or glutamine abolished or greatly decreased levels of methylation of lysine 20 of H4 by SET8. Interestingly, mutation of lysine 16 to alanine, arginine, glutamine, or methionine did not affect methylation of lysine 20 by the SET domain. Mass spectrometric analysis of synthesized H4 N-terminal peptides modified by SET8 showed that SET8 selectively mono-methylates lysine 20 of H4. Taken together, our results suggested that the coordination between the amino acid sequence RHRK20VLRDN and the SET domain of SET8 determines the substrate specificity and multiplicity of methylation of lysine 20 of H4.     

Structure of the Neurospora SET domain protein DIM-5, a histone H3 lysine methyltransferase

AdoMet-dependent methylation of histones is part of the "histone code" that can profoundly influence gene expression. We describe the crystal structure of Neurospora DIM-5, a histone H3 lysine 9 methyltranferase (HKMT), determined at 1.98 A resolution, as well as results of biochemical characterization and site-directed mutagenesis of key residues. This SET domain protein bears no structural similarity to previously characterized AdoMet-dependent methyltransferases but includes notable features such as a triangular Zn3Cys9 zinc cluster in the pre-SET domain and a AdoMet binding site in the SET domain essential for methyl transfer. The structure suggests a mechanism for the methylation reaction and provides the structural basis for functional characterization of the HKMT family and the SET domain.     

SET domains and histone methylation

The realisation that SET domains, which are found in numerous proteins involved in chromatin regulation, catalyse the methylation of lysine residues has led to intense interest in their cellular, biochemical and structural properties. The structures of five SET domain proteins have been reported over the past year. SET domains possess a novel fold, and use adjacent domains for both structural stabilisation and the completion of their active sites. The cofactor S-adenosyl-L-methionine and peptide substrates bind on opposite faces of the SET domain. Remarkably, the sidechain of the target lysine approaches the transferred methyl group through a narrow channel that passes through the middle of the domain.     

Kinetic manifestation of processivity during multiple methylations catalyzed by SET domain protein methyltransferases

Processive versus distributive methyl group transfer was assessed for pea Rubisco large subunit methyltransferase, a SET domain protein lysine methyltransferase catalyzing the formation of trimethyllysine-14 in the large subunit of Rubisco. Catalytically competent complexes between an immobilized form of des(methyl) Rubisco and Rubisco large subunit methyltransferase were used to demonstrate enzyme release that was co-incident with and dependent on formation of trimethyllysine. Catalytic rate constants determined for formation of trimethyllysine were considerably lower ( approximately 10-fold) than rate constants determined for total radiolabel incorporation from [3H-methyl]-S-adenosylmethionine. Double-reciprocal velocity plots under catalytic conditions favoring monomethyllysine indicated a random or ordered reaction mechanism, while conditions favoring trimethyllysine suggested a hybrid ping-pong mechanism. These results were compared with double-reciprocal velocity plots and product analyses obtained for HsSET7/9 (a monomethyltransferase) and SpCLR4 (a dimethyltransferase) and suggest a predictive ability of double-reciprocal velocity plots for single versus multiple methyl group transfers by SET domain protein lysine methyltransferases. A model is proposed for SET domain protein lysine methyltransferases in which initial binding of polypeptide substrate and S-adenosylmethionine is random, with polypeptide binding followed by deprotonation of the epsilon-amine of the target lysyl residue and subsequent methylation. Following methyl group transfer, S-adenosylhomocysteine and monomethylated polypeptide dissociate from monomethyltransferases, but di- and trimethyltransferases begin a successive and catalytically obligatory deprotonation of enzyme-bound methylated lysyl intermediates, which along with binding and release of S-adenosylmethionine and S-adenosylhomocysteine is manifested as a hybrid ping-pong-like reaction mechanism.     

Dominant alleles identify SET domain residues required for histone methyltransferase of Polycomb repressive complex 2

Polycomb gene silencing requires histone methyltransferase activity of Polycomb repressive complex 2 (PRC2), which methylates lysine 27 of histone H3. Information on how PRC2 works is limited by lack of structural data on the catalytic subunit, Enhancer of zeste (E(Z)), and the paucity of E(z) mutant alleles that alter its SET domain. Here we analyze missense alleles of Drosophila E(z), selected for molecular study because of their dominant genetic effects. Four missense alleles identify key E(Z) SET domain residues, and a fifth is located in the adjacent CXC domain. Analysis of mutant PRC2 complexes in vitro, and H3-K27 methylation in vivo, shows that each SET domain mutation disrupts PRC2 histone methyltransferase. Based on known SET domain structures, the mutations likely affect either the lysine-substrate binding pocket, the binding site for the adenosylmethionine methyl donor, or a critical tyrosine predicted to interact with the substrate lysine epsilon-amino group. In contrast, the CXC mutant retains catalytic activity, Lys-27 specificity, and trimethylation capacity. Deletion analysis also reveals a functional requirement for a conserved E(Z) domain N-terminal to CXC and SET. These results identify critical SET domain residues needed for PRC2 enzyme function, and they also emphasize functional inputs from outside the SET domain.     

Structural basis for the methylation site specificity of SET7/9

Human SET7/9 is a protein lysine methyltransferase (PKMT) that methylates histone H3, the tumor suppressor p53 and the TBP-associated factor TAF10. To elucidate the determinants of its substrate specificity, we have solved the enzyme's structure bound to a TAF10 peptide and examined its ability to methylate histone H3, TAF10 and p53 substrates bearing either mutations or covalent modifications within their respective methylation sites. Collectively, our data reveal that SET7/9 recognizes a conserved K/R-S/T/A motif preceding the lysine substrate and has a propensity to bind aspartates and asparagines on the C-terminal side of the lysine target. We then used a sequence-based approach with this motif to identify novel substrates for this PKMT. Among the putative targets is TAF7, which is methylated at Lys5 by the enzyme in vitro. These results demonstrate the predictive value of the consensus motif in identifying novel substrates for SET7/9.     

Crystal structure and functional analysis of the histone methyltransferase SET7/9

Methylation of lysine residues in the N-terminal tails of histones is thought to represent an important component of the mechanism that regulates chromatin structure. The evolutionarily conserved SET domain occurs in most proteins known to possess histone lysine methyltransferase activity. We present here the crystal structure of a large fragment of human SET7/9 that contains a N-terminal beta-sheet domain as well as the conserved SET domain. Mutagenesis identifies two residues in the C terminus of the protein that appear essential for catalytic activity toward lysine-4 of histone H3. Furthermore, we show how the cofactor AdoMet binds to this domain and present biochemical data supporting the role of invariant residues in catalysis, binding of AdoMet, and interactions with the peptide substrate.     

Mechanism of multiple lysine methylation by the SET domain enzyme Rubisco LSMT

SET domain protein methyltransferases catalyze the transfer of methyl groups from the cofactor S-adenosylmethionine (AdoMet) to specific lysine residues of protein substrates, such as the N-terminal tails of histones H3 and H4 and the large subunit of the Rubisco holoenzyme complex. The crystal structures of pea Rubisco large subunit methyltransferase (LSMT) in ternary complexes with either lysine or epsilon-N-methyllysine (MeLys) and the product S-adenosylhomocysteine (AdoHcy) were determined to resolutions of 2.65 and 2.55 A, respectively. The zeta-methyl group of MeLys is bound to the enzyme via carbon-oxygen hydrogen bonds that play a key role in catalysis. The methyl donor and acceptor are aligned in a linear geometry for S(N)2 nucleophilic transfer of the methyl group during catalysis. Differences in hydrogen bonding between the MeLys epsilon-amino group and Rubisco LSMT and SET7/9 explain why Rubisco LSMT generates multiply methylated Lys, wheras SET7/9 generates only MeLys.     

Structural and biochemical studies of human lysine methyltransferase Smyd3 reveal the important functional roles of its post-SET and TPR domains and the regulation of its activity by DNA binding

The SET- and MYND-domain containing (Smyd) proteins constitute a special subfamily of the SET-containing lysine methyltransferases. Here we present the structure of full-length human Smyd3 in complex with S-adenosyl-l-homocysteine at 2.8 Å resolution. Smyd3 affords the first example that other region(s) besides the SET domain and its flanking regions participate in the formation of the active site. Structural analysis shows that the previously uncharacterized C-terminal domain of Smyd3 contains a tetratrico-peptide repeat (TPR) domain which together with the SET and post-SET domains forms a deep, narrow substrate binding pocket. Our data demonstrate the important roles of both TPR and post-SET domains in the histone lysine methyltransferase (HKMT) activity of Smyd3, and show that the hydroxyl group of Tyr239 is critical for the enzymatic activity. The characteristic MYND domain is located nearby to the substrate binding pocket and exhibits a largely positively charged surface. Further biochemical assays show that DNA binding of Smyd3 can stimulate its HKMT activity and the process may be mediated via the MYND domain through direct DNA binding.     

A novel SET domain methyltransferase in yeast: Rkm2-dependent trimethylation of ribosomal protein L12ab at lysine 10

The ribosomal protein L12ab (Rpl12ab) in Saccharomyces cerevisiae is modified by methylation at both arginine and lysine residues. Although the enzyme responsible for the modification reaction at arginine 66 has been identified (Rmt2), the enzyme(s) responsible for the lysine modification(s) has not been found, and the site(s) of methylation has not been determined. Here we demonstrate, using a combination of mass spectrometry and labeling assays, that the yeast gene YDR198c encodes the enzyme responsible for the predominant epsilon-trimethylation at lysine 10 in Rpl12ab. An additional site of predominant epsilon-dimethylation is observed at lysine 3; the enzyme catalyzing this modification is not known. The YDR198c gene encodes a SET domain similar to that of the Rkm1 enzyme responsible for modifying Rpl23ab, and we have now designated the YDR198c gene product as Rkm2 (ribosomal lysine methyltransferase 2). The effect of the loss of the enzyme on ribosomal complex stability was studied by polysomal fractionation. However, no difference was observed between the Deltarkm2 deletion strain and its parent wild type strain. With the identification of this enzyme, it appears that the 12 SET domain family members in yeast can now be divided into two subfamilies based on function and amino acid sequence identity. One branch includes enzymes that modify histones, including Set1 and Set2; the other branch includes Rkm1, Rkm2, and Ctm1, the cytochrome c methyltransferase. These studies suggest that the remaining seven SET domain proteins may also be lysine methyltransferases.     

Structural Context of Disease-Associated Mutations and Putative Mechanism of Autoinhibition Revealed by X-Ray Crystallographic Analysis of the EZH2-SET Domain

The enhancer-of-zeste homolog 2 (EZH2) gene product is an 87 kDa polycomb group (PcG) protein containing a C-terminal methyltransferase SET domain. EZH2, along with binding partners, i.e., EED and SUZ12, upon which it is dependent for activity forms the core of the polycomb repressive complex 2 (PRC2). PRC2 regulates gene silencing by catalyzing the methylation of histone H3 at lysine 27. Both overexpression and mutation of EZH2 are associated with the incidence and aggressiveness of various cancers. The novel crystal structure of the SET domain was determined in order to understand disease-associated EZH2 mutations and derive an explanation for its inactivity independent of complex formation. The 2.00 Å crystal structure reveals that, in its uncomplexed form, the EZH2 C-terminus folds back into the active site blocking engagement with substrate. Furthermore, the S-adenosyl-L-methionine (SAM) binding pocket observed in the crystal structure of homologous SET domains is notably absent. This suggests that a conformational change in the EZH2 SET domain, dependent upon complex formation, must take place for cofactor and substrate binding activities to be recapitulated. In addition, the data provide a structural context for clinically significant mutations found in the EZH2 SET domain.     

Structure of the Catalytic Domain of EZH2 Reveals Conformational Plasticity in Cofactor and Substrate Binding Sites and Explains Oncogenic Mutations

Polycomb repressive complex 2 (PRC2) is an important regulator of cellular differentiation and cell type identity. Overexpression or activating mutations of EZH2, the catalytic component of the PRC2 complex, are linked to hyper-trimethylation of lysine 27 of histone H3 (H3K27me3) in many cancers. Potent EZH2 inhibitors that reduce levels of H3K27me3 kill mutant lymphoma cells and are efficacious in a mouse xenograft model of malignant rhabdoid tumors. Unlike most SET domain methyltransferases, EZH2 requires PRC2 components, SUZ12 and EED, for activity, but the mechanism by which catalysis is promoted in the PRC2 complex is unknown. We solved the 2.0 Å crystal structure of the EZH2 methyltransferase domain revealing that most of the canonical structural features of SET domain methyltransferase structures are conserved. The site of methyl transfer is in a catalytically competent state, and the structure clarifies the structural mechanism underlying oncogenic hyper-trimethylation of H3K27 in tumors harboring mutations at Y641 or A677. On the other hand, the I-SET and post-SET domains occupy atypical positions relative to the core SET domain resulting in incomplete formation of the cofactor binding site and occlusion of the substrate binding groove. A novel CXC domain N-terminal to the SET domain may contribute to the apparent inactive conformation. We propose that protein interactions within the PRC2 complex modulate the trajectory of the post-SET and I-SET domains of EZH2 in favor of a catalytically competent conformation.     

Yeast ribosomal/cytochrome c SET domain methyltransferase subfamily: identification of Rpl23ab methylation sites and recognition motifs

Ribosomal protein L23ab is specifically dimethylated at two distinct sites by the SET domain-containing enzyme Rkm1 in the yeast Saccharomyces cerevisiae. Using liquid column chromatography with electrospray-ionization mass spectrometry, we determined that Rpl23ab purified from the Deltarkm1 deletion strain demonstrated a loss in mass of approximately 56 Da when compared with Rpl23ab purified from the wild type strain. When Rpl23ab was proteolyzed, using proteinase ArgC or CNBr, and the peptides derived were analyzed by tandem mass spectrometry, no sites of methylation were found in Rpl23ab purified from the Deltarkm1 deletion strain, whereas two sites of dimethylation were observed in the wild type strain at lysine residues 105 and 109. We show that both Rpl23a and Rpl23b are expressed and methylated in vivo in yeast. Using polysomal fractionation, we demonstrate that the deletion of RKM1 has no effect on ribosomal complex formation. Comparison of SET domain methyltransferase substrates in yeast reveal sequence similarities around the lysine methylation sites and suggest that an (Asn/Pro)-Pro-Lys consensus sequence may be a target for methylation by subfamily 2 SET domain methyltransferases. Finally, we show the presence of Rkm1 homologs in fungi, plants, and mammals including humans.