Protein Methyltransferases: A Distinct, Diverse, and Dynamic Family of

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Protein methyltransferases: A distinct, diverse and dynamic family of enzymes P. Ann Boriack-Sjodin, and Kerren K Swinger Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.5b01129 • Publication Date (Web): 10 Dec 2015 Downloaded from http://pubs.acs.org on December 15, 2015

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Protein methyltransferases: A distinct, diverse and dynamic family of enzymes P. Ann Boriack-Sjodin and Kerren K. Swinger Epizyme, Inc. 400 Technology Square, Cambridge, MA 02139 Address correspondence to P. Ann Boriack-Sjodin. Email: [email protected]

Abstract Methyltransferase proteins are a superfamily of enzymes that add one or more methyl groups to substrates that include protein, DNA, RNA and small molecules. The subset of proteins that act upon arginine and lysine side chains are characterized as epigenetic targets due to their activity on histone molecules and their ability to affect transcriptional regulation. However, it is now clear that these enzymes target other protein substrates as well, greatly expanding their potential impact on normal and disease biology. Protein methyltransferases are well characterized structurally. In addition to revealing the overall architecture of the subfamilies of enzymes, structures of complexes with substrates and ligands have enabled detailed analysis of biochemical mechanism, substrate recognition, and design of potent and selective inhibitors. This review focuses on how knowledge gained from structural studies has impacted the understanding of this large class of epigenetic enzymes.

Introduction Since their discovery less than two decades ago, methyltransferases have developed into one of the largest classes of epigenetic enzymes and continue to be an active area of research for industry and academia alike. More than half of the known epigenetic enzymes add or remove methyl groups from protein, DNA, or RNA substrates, resulting in changes in transcriptional

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regulation (1). The largest enzyme class is the writers that act upon protein substrates, protein methyltransferases (PMTs). Initially labeled as histone methyltransferases, there is a growing understanding that histone molecules are only one of many potential protein targets within the cell (2). Ironically, this knowledge greatly expands yet also complicates the biology of protein methylation. It is known that dysregulation of epigenetic targets can lead to pathological changes that result in human disease (3), hence protein methyltransferases are active targets for therapeutic intervention. Inhibitors for two PMTs, DOT1L and EZH2, have reached the clinic, and efforts by pharmaceutical and biotechnology companies are reported for several other family members. Therefore, understanding the structure and function of these enzymes may have potential implications for drug discovery efforts as well as expand knowledge of the biology of this enzyme class. Protein methyltransferases can be broadly differentiated based on the target amino acid of the methylation reaction. This review focuses on the lysine and arginine methyltransferases. Phylogenic trees for these PMTs have been designed (4-6), dividing the methyltransferases into smaller subfamilies based on chemogenic analysis, and this review will feature several of these subfamilies in detail (Figure 1). Although the catalytic domains of lysine or arginine PMTs share common sequence and structural features, there is significant diversity in sequence and quaternary structure between the enzymes. Some protein methyltransferases exist as globular proteins, such as the SMYD (SET and MYND domain containing) family (7), while other enzymes contain multiple, distinct domains. For example, in addition to the methyltransferase catalytic domain, NSD (nuclear receptor SET domain-containing) family member WHSC1L1 contains PHD (plant homeodomain) fingers, PWWP (Pro-Trp-Trp-Pro motif) domains, and an NSD specific C5HCH domain (a cysteine-rich domain) (8), and structures have been solved for


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the catalytic domain (Tempel et al; PDB ID 4YZ8), a PWWP domain (Qin I., PDB ID 4RXJ) and the tandem PHD5-C5HCH domains (9). Although many proteins are active as individual full-length or truncated enzymes, PMTs are often found in multi-protein complexes and a few, such as EZH2 (10), are only functional within these larger units. Knowledge of the environment in which a PMT is found in vivo is important when analyzing structural and functional data obtained in vitro, particularly for truncated enzymes. Structural biology efforts have been extremely productive for this class of enzymes, as structures of PMT catalytic domains are known for more than half of PMT family members (6). A subset of enzymes has been crystallized in ternary complexes containing both peptide and nucleotide substrates or analogs, providing a basis for understanding substrate specificity. Additionally, there is active research to develop tool compounds to understand the complex biology of PMTs (11, 12), and structural biology is playing a key role in developing many of these potent and selective reagents. In this review, structural biology efforts for several subfamilies of PMTs will be examined detailing the diversity within the enzyme family, highlighting the role protein dynamics plays for many of these targets, and specifying the continuing role structure will play in understanding the biochemistry and biology of these complex enzymes.

Domain structure of PMTs With one exception, lysine methyltransferases (PKMTs) contain a catalytic SET (Su(var), E(z) and Trithorax) domain defined by specific amino acid motifs (ELxF/YDY and RFINHxCxPN where x is any amino acid) and a pseudo-knot structure (13) (Figure 2A). Although the SET domain contains all residues important for catalysis, I-SET (Immunoglobulin-


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SET) and post-SET domains are also found in all SET-containing protein methyltransferases. These additional domains contribute to the peptide binding pocket and in many cases to the Sadenosylmethionine (SAM ) binding site as well (13). DOT1L is a structurally unique lysine methyltransferase (Figure 2B), as described below. Arginine methyltransferases (PRMTs) are structurally distinct from SET domaincontaining PKMTs. The catalytic core of these proteins contains three domains (Figure 2C). The first is an MTase domain (methyltransferase domain) that is similar to the Rossmann fold and includes all the residues that contribute to the SAM binding site. This domain is structurally conserved (14). The second domain is a β-barrel unique to PRMT enzymes, and the third is a dimerization domain (14). Substrate peptides bind in a pocket located at the interface between the MTase domain and the β-barrel (15-18). Although PRMTs share sequence homology with other methyltransferase enzymes (5), only arginine methyltransferases structures will be discussed.

PMT mechanism and specificity To appreciate PMT structure one must also understand the mechanism of these enzymes. All PMTs require two substrates for enzyme catalysis, SAM and the targeted residue of methylation. SAM binds to the protein and donates a methyl group to the lysine or arginine side chain of the protein substrate using an SN2-based transfer reaction producing Sadenosylhomocysteine (SAH) in the process. PKMT enzymes bind lysine residues in a narrow hydrophobic channel within the SET domain and orient the terminal ε-amine using carbonoxygen hydrogen bonding (19). Water molecules play a key role in the deprotonation event for PKMT enzymes (20). PRMT-substrate enzyme complexes (15-18) do not have a conserved


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water molecule for deprotonation of the arginine side chain. The higher pKa of arginine may necessitate a different proton transfer mechanism (21). It is also possible that deprotonation of the guanidino moiety is not essential prior to transfer of the methyl group from SAM to substrate (22). Lysine can exist in four possible methylation states: unmodified or having one, two, or three methyl groups covalently bound (Figure 3A). Arginine can also exist in several methylation states including unmodified, monomethylated or dimethylated; dimethylated arginine residues can occur in either symmetric (1 methyl group per terminal nitrogen atom) or asymmetric (2 methyl groups on a single terminal nitrogen atom) geometry (Figure 3B). The addition of more than one methyl group to protein residues can be performed by a single enzyme or may be performed by different enzymes. For example, wild type EZH2 can produce mono-, di- and tri-methylated lysine at position 27 of histone H3 (H3K27) with differing catalytic efficiencies for each substrate, and active site mutations can alter these kinetic profiles (23). In contrast, H3K36 is trimethylated by two different enzymes; mono- and dimethylation occurs through the NSD family (24), while trimethylation is performed by SETD2 (25). Some methylation sites have unique methyltransferase writers, such as H3K79 methylation by DOT1L (26), while H3K9 is a substrate for no fewer than eight human methyltransferases (4), and p53 is a target for at least five lysine methyltransferases (27).

Conversely, individual PMTs can have

multiple protein substrates. SETD7 has at least 11 non-histone substrates characterized by in vitro or in vivo methods (27). The interplay between enzymes and substrates creates opportunities for structural biology and biochemistry efforts to develop and test hypotheses regarding substrate specificities and kinetic mechanisms.


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SETD7: A model methyltransferase enzyme SETD7 was the first mammalian PKMT solved by protein crystallography (28, 29) and together with the structures of Neurospora crassa DIM-5 (30), Schizosaccharomyces pombe Clr4 (31), and garden pea Rubisco LSMT (32) defined the three-dimensional SET domain architecture and demonstrated the SET methyltransferase fold was evolutionarily conserved across a variety of species. To date, nearly 30 structures of wild type and mutant SETD7 proteins have been deposited in public databases, more than any other individual PMT. In addition to structures of SETD7 bound to SAM and SAH, complexes with various substrate peptides or inhibitors occupying either SAM or peptide binding sites have been solved. Many of the structures solved for SETD7 were generated because the enzyme has been used as a model system to illuminate structural aspects of methyltransferase biochemistry. For example, active site mutants of SETD7 are known that alter product specificity from a monomethylase to an enzyme that can catalyze di- and trimethylation (33) . Structural characterization of these mutants in complex with peptides differing in methylation states highlighted the important role active site water molecules perform in orienting the lysine ε nitrogen with increasing numbers of methyl groups prior to enzyme catalysis (34). Additional structural and biochemical studies of active site mutants of SETD7 showed CH---O hydrogen bonds constrict the motion of the SAM methyl group and are critical to high affinity SAM binding and transition-state stabilization during catalysis (35). As stated previously, SETD7 methylates multiple substrate protein and several structures of SETD7-peptide complexes are known (histone H3, TAF10, p53, ERα and DNMT1). Analysis of the binding interactions lead to the hypothesis of a consensus motif (27). However, more recent analysis of SETD7 substrate sequences reveals sequences outside the consensus sequence are also substrates (36). This result


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may indicate that interactions beyond the SET domain play an important role in substrate specificity (27) and provides another opportunity for structural biology efforts to contribute to the understanding of substrate recognition for this model enzyme.

Suv39/EHMT: A model methyltransferase family The Suv39/EHMT family of PKMTs includes EHMT1/2 (GLP/G9a), SUV39H1/2, SETDB1/2, and SETMAR. With the exception of SETDB1/2, the sequence of which is unique due to a large insertion domain within the SET domain, all family members have been structurally characterized (6). This group was the first PMT subfamily to have apo, peptidebound and inhibitor-bound structures, making these enzymes an early model for understanding substrate and inhibitor specificity and enabling structure-guided design of probe molecules. H3K9 is a substrate of several PMT enzymes, including all members of the Suv39/EHMT family except SETMAR (4). Crystal structures of human EHMT1, EHMT2 and Suv39H2 were compared to each other and to other H3K9 enzymes including human PRDM2 to determine similarities between protein structures for enzymes with the same substrate (4) The comparisons showed conservation in I-SET conformation and variability in post-SET domain dependent on the presence or absence of the substrate peptide, indicating a pre-formed platform may be necessary for substrate binding but that protein dynamics were also important to binding and recognition (4). However, sequence differences in the substrate ablated binding for one methyltransferase while only mildly affecting another, indicating selectivity mechanisms may not be conserved even for enzymes with the same substrate (4). Structural biology has played a pivotal role in the development of tool compounds for the EHMT enzymes. BIX-01294 (37) was the first inhibitor of a PMT molecule whose structure was


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documented in the literature (38). Comparison of the GLP-BIX01294 structure to that of GLP bound to an H3K9 peptide (4) showed the compound bound in the peptide binding site and revealed plasticity in the side chain conformation of Arg1214 dependent on the identity of the ligand. The structure also revealed the importance of the αZ helix to the specificity of the compound for EHMT1 and EHMT2 over other PMT enzymes tested. Interestingly, BIX-01294 did not engage the lysine channel, and subsequent work within this scaffold showed targeting this pocket, in addition to other interactions, resulted in dramatic increases in potency (39, 40). Additional efforts, often guided by structure-based design, have resulted in numerous scaffolds suitable as in vitro and in vivo probe molecules (39, 41-44) and enabled testing of biological hypotheses for these enzymes.

NSD and related sub-family members: A dynamic class of enzymes The NSD family of enzymes contains three members: NSD1, WHSC1 (NSD2/MMSET) and WHSC1L1 (NSD3). Ranging in size from 1365 amino acids (WHSC1) to 2696 amino acids (NSD1), all family members contain a SET domain near the C-terminus followed by a PHD domain, as well as containing three additional PHD and two PWWP domains (25). Each of these proteins may play an important role in select cancers. WHSC1 has been demonstrated as a driver in multiple myeloma containing the t(4:14) chromosomal translocation, while NUP98NSD1 and NUP98-WHSC1L1 fusion proteins have been described in acute myeloid leukemias (45). Overexpression of these proteins has also been seen in different cancer cell lines. Therefore, there is much interest in understanding the structure and catalytic mechanism of this family of enzymes in both academia and industry.


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To date, NSD1 (Figure 4A) and WHSC1L1 (Figure 4B) each have only a single SAMbound structure of the SET domain for each enzyme in the public domain, while the WHSC1 SET domain remains structurally uncharacterized. However, ASH1L and SETD2 are closely related proteins on the phylogenic trees (Figure 1), and additional structural and biochemical studies on these proteins have been recently documented. Examination of these proteins provides key insights into the role protein dynamics may have in enzymatic turnover within the NSD family. The structures of NSD1 (46), SETD2 (47), and ASH1L (48) reveal the post-SET loops of these enzymes occupy an auto-inhibitory conformation which sterically blocks substrate from accessing the lysine channel (Figure 4C). However, additional structures of SETD2 and ASH1L reveal this same loop can adopt a number of conformations under different conditions. A structure of SETD2 bound to SFG-Pr, a nucleotide analog with an extended group in the lysine pocket, results in a flipped conformation of the loop and an open conformation of the protein that may be accessible to peptide substrates (46) (Figure 4C). Select point mutants in the autoinhibitory loop of ASH1L had varying effects on catalytic activity of the enzyme, and several different conformations of this loop and the I-SET loop were documented (49). Both studies postulated that the conformation of the loop is important for substrate recognition and acts as a regulatory feature beyond a a loop-open or loop-closed binary gating mechanism.

Taken

together, these structures reveal the auto-inhibitory loop to be a conformationally dynamic region of this family of proteins that is critical to the catalytic mechanism. This flexibility may be of importance for those interested in drug discovery efforts. Interestingly, the recent structure of the SET domain of WHSC1L1 (Tempel et al; PDB ID 4YZ8) does not include electron density for residues 1264-1268 (Figure 4A), presumably because these residues exist in multiple conformations or are disordered. As this is the only available structure for the SET domain of


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WHSC1L1, additional studies will be needed to determine whether this flexibility is an inherent feature of the protein or a consequence of the truncated protein construct that was characterized crystallographically.

SMYD family: PMTs with deep pockets The SMYD proteins comprise a family of five soluble enzymes that are known to target histone and non-histone substrates (7). All SMYD family members contain a MYND domain, a zinc finger motif known to mediate protein-protein interactions with proline-rich sequences (50). Crystal structures of the full-length proteins are known for three of the five family members: SMYD1 (51), SMYD2 (52-54), and SMYD3 (55-57)(Lam et al, PDB ID 3MEK). These molecules share a bilobal architecture; the N-terminal lobe contains the SET, I-Set, post-SET and MYND domains, while the α-helical C-terminal domain shows similarities to TPR domains despite disparate sequences (7). The most structurally dissimilar molecules in the family are as yet unsolved. SMYD4 contains additional TPR domains at the N-terminus and is nearly twice the size of the other SMYD molecules, while SMYD5 has a unique C-terminal sequence that is unrelated to the known C-terminal domains (7). The interface of the N- and C-terminal lobes forms large, deep binding sites for protein substrates. Structural diversity between the molecules is defined by both the sequence and orientation of the C-terminal lobes, resulting in different surface topologies for SMYD1, 2, and 3 peptide binding sites (Figure 5A-C). In all structures, the nucleotide is bound in the N-terminal domain, with conserved residues forming a narrow channel for the lysine substrate before opening up to large, solvent-filled regions (7). Structures of p53 (52, 54) and ERα (58) peptides have been solved in complex with SMYD2. Both peptides follow a similar U-shaped


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conformation and are superimposable by the substrate lysine and surrounding residues, but diverge in position and interactions at residues further from the site of methylation. Comparison of the similarities and differences in recognition motifs for different substrates will provide a greater understanding of substrate specificity of the SMYD enzymes. Therefore, additional structures of SMYD2-peptide complexes and substrate complexes for other SMYD enzymes are desired. SMYD2 and SMYD3 methylate targets known to be important in cancer biology, potentially affecting signal transduction pathways. SMYD3 methylates MAP3K2 and has been shown to promote Ras-mediated cancer progression (59). The tumor suppressor p53 is a substrate for SMYD2 and methylation by SMYD2 was shown to repress its function (60). Additionally, overexpression of SMYD2 and 3 has been documented in several cancers (reviewed in (7)). Therefore, there is significant interest in the development of compounds to specifically inhibit each SMYD enzyme. Although the peptide binding sites are relatively large, potent small molecule inhibitors have been found for both SMYD2 and SMYD3. Two distinct chemical series, defined by the chemical moiety found in the lysine channel, have been published for SMYD2: a benzooxazinone series (AZ505 (52), A-893 (61)), and a pyrrolidine series (LLY507 (62)). Despite differences in chemical structure, both series bind to SMYD2 in a similar fashion, engaging the lysine channel and clasping the surface of the protein while engaging two hydrophobic pockets. In contrast, the potent SMYD3 inhibitor, EPZ030456, exits the lysine channel and extends directly into the solvent filled cavity of the enzyme (63). The structures show that at least two distinct binding modes can lead to potent and selective inhibitionof the SMYD family (Figure 5D).


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PRDMs: Evolutionary cousins of SET-domain containing enzymes The PRDM family is defined by the presence of an N-terminal PR (PRDI-BF1 and RIZ1 homology) domain. Despite low (20-30%) sequence identity, PR domains are structurally related to SET domains and may be evolutionarily derived from these enzymes (64). Interestingly, although PR domains lack one of the key sequence motifs (NHxC) that define the SET domain (65) and are important to catalytic function (66), only select members of the family (PRDM2/RIZ1, PRDM8, PRDM9) have been shown to methylate histones (67-71). Studies with other PRDM enzymes have been more enigmatic. PRDM3 and PRDM16 were reported to have methyltransferase activity (68), but subsequent work with a PR domain mutant of PRDM16 also showed methyltransferase activity, potentially due to association of PRDM16 with EHMT1 (72). The reasons why some PRDM family members are not active methyltransferases is currently not known, but catalytically inactive family members can contribute to chromatin structure remodulation through the recruitment of modifying enzymes or complexes, including SET domain containing PMTs (65, 73). The PR domain of PRDM2 was the first to be structurally characterized and was solved by both NMR and x-ray crystallography (4, 74). The crystal structures, solved in the absence of peptide or nucleotide substrates, confirmed that the PR domain retained the predicted SET domain fold (Figure 6A), while the NMR structure showed high mobility in the Post-SET domain, a sequence involved in substrate binding. Additionally, high dissociation constants were found for the H3 peptide (0.7 mM) and product SAH (>10 mM) for this catalytically active PRDM (74). To date, several crystal structures of PR domains of PRDM proteins have been solved, but only one, murine PRDM9, has been solved with substrates bound (71). Despite the lack of conserved sequences, the SAH molecule is bound to the enzyme in a similar way in


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PRDM9 as in SET domain proteins. However, sequence differences between PRDM molecules with catalytic activity indicate the binding mode of PRDM9 with nucleotide may not be conserved within the PRDM family (71). Comparison of substrate-bound and apo forms of PRDM9 also indicate the post-SET domain undergoes conformational changes in the absence of substrates and adopts an auto-inhibited conformation (Figure 6B). Thus, while PRDM enzymes exhibit many of the same characteristics seen in their SET domain containing relatives, additional structural studies of catalytically active PRDM molecules with substrates bound may be beneficial in understanding the structure-activity relationships within this enigmatic subfamily of PMT enzymes.

Dot1L: A unique lysine methyltransferase As previously noted, DOT1L is a structurally unique enzyme within the PKMT superfamily. Although it is active against H3K79 and is therefore a lysine methyltransferase, early sequence analysis showed characteristics similar to that of the arginine methyltransferases (75) rather than SET domain-containing molecules. The structure of the catalytic domain was first solved in 2003. The structure revealed an elongated molecule with two domains distinct from SET-containing proteins and confirmed similarity to previously solved non-SET domain containing methyltransferases (76). The SAM binding site of DOT1L is contained in the Cterminal α/β domain (Figure 3B, 7A). Subsequent comparisons of the conformation of SAM in DOT1L overlaid well with PRMTs and was dissimilar to SAM conformations in SET domain containing proteins (5). Recent structural analyses have revealed that DOT1L is a dynamic enzyme that accesses a number of conformational states. When bound to iodotubercidin, a small nucleotide analog


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with µM potency, the activation loop adopts an open conformation such that the adenine ring is more solvent exposed than it is in the SAM-bound conformation (77) (Figure 7B). Crystal structures of DOT1L bound to several SAM-competitive inhibitors (77-81) together with hydrogen/deuterium exchange experiments (77) indicate that loops in and near the SAM binding site adopt several different discrete conformations or can be structurally disordered when bound to different chemical classes. Some potent and selective inhibitors of DOT1L induce a pocket within the enzyme that is not present in the SAM, SAH or iodotubercidin structures (Figure 7CD). Significant conformational changes to the substrate-binding loop and movements of side chains occur when these inhibitors bind, burying a hydrophobic portion of the compounds within the protein core (77-79). One of these compounds, pinometostat (EPZ-5676), has been extensively characterized biochemically and structurally. Pinometostat has a Ki of 80 pM and a residence time on DOT1L protein of more than 24 hours (79). The conformational changes that are required for this inhibitor to bind likely contribute to its potency, selectivity, and residence time (82). It is possible that the protein dynamics seen in DOT1L structures are important for substrate recognition and binding or enzyme turnover. For example, DOT1L is only active in vitro on nucleosomes and ubiquitinylation of H2B has been demonstrated to stimulate activity (83). Characterization of DOT1L substrate complexes would help to elucidate the structural underpinnings of substrate-based effects on activity. This question will require additional experimental efforts, as no structure of a DOT1L-substrate complex is currently available.

Protein arginine methyltransferases (PRMTs): A subfamily with similarity and diversity


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Mammalian PRMT enzymes are divided into separate sub-classes of enzymes based on the type of methylation performed on the terminal nitrogen atoms of the arginine residue: monomethylation, symmetric dimethylation or asymmetric dimethylation (84). Type I enzymes (PRMT1, 2, 3, 6, 8 and 4 (also known as CARM1)) produce asymmetric dimethylation products after first producing the monomethylated arginine product. Type II enzymes (PRMT5, 7) produce symmetrically methylated arginine residues after production of the monomethylated state (Figure 2B). The first structure of SAH-bound PRMT3 solved in 2000 (85) documented the domain structure common to PRMT enzymes (Figure 3C), identified the arginine binding site, and enabled postulation of a catalytic mechanism for the PRMT family. To date, several structures of mammalian PRMTs have been solved (Figure 8) and knowledge gained from the sum of these structures allows insights into this subfamily of PMTs beyond the gross architecture of the catalytic core. CARM1 structures solved with and without nucleotide show structural rearrangement occurs upon binding of the substrate (86, 87), and highlight the role protein dynamics likely play in substrate turnover. In addition, domains external to the catalytic domain can contribute to the various protein-protein interactions important for activity and biology. Two examples include the structure of PRMT5 bound to MEP50 (16, 88) and the structure of an isolated construct of the N-terminus of CARM1 revealing a PH domain architecture (87). The structure of C. elegans (89) and murine (90) PRMT7 revealed a tandem repeat of the PRMT core likely achieved by gene duplication, although only one of the domains had catalytic activity. Interestingly, the structure of PRMT7 from a more primitive organism Trypanosoma brucei did not show this gene duplication (17).


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The structure of PRMT1 was the first arginine methyltransferase to provide evidence of the substrate binding mode. Although structural rearrangement was observed in the PRMT1 binding site, an arginine residue was seen engaging in the substrate pocket (15). In addition, evidence of multiple binding sites for the protein substrate external to the arginine channel was visible, though multiple binding modes obscured any analysis of substrate recognition motifs. The structure of PRMT5-MEP50 complexed with a histone H4-derived peptide first detailed the specific interactions made between the peptide substrate and the PRMT enzyme beyond the arginine pocket (16). More recent structures of human CARM1 (18) and of Trypanosoma brucei PRMT7 (17) with peptide substrates have underscored the engagement of backbone rather than side chain interactions beyond the arginine residue, which may be thematic for enzymes with multiple protein substrates. Additionally, these structures show the absence of large conformational changes upon peptide binding, in contrast to select PKMT enzymes. As was seen in SET domain containing enzymes, structure has played a significant role in the development of inhibitors for the PRMT enzymes. Type I enzymes are structurally similar, and inhibitors that target multiple PRMTs have been documented (91, 92). Even so, selectivity is possible within the family, as was shown with both CARM1-selective (Figure 8B) and PRMT6-selective compounds (Figure 8D) (91, 93). A recent potent and selective in vivo tool compound for PRMT5 (Figure 8C) was published revealing a π-cation interaction between the tetrahydroisoquinoline headgroup and the SAM substrate unique in PRMT inhibitors to date (94). Selective inhibitors for PRMT3 have been achieved through an allosteric mechanism (95, 96). In this case the compounds bind the dimer interface (Figure 8E) changing the conformations of helix αY and causing disorder in helix αX, reminiscent of the disorder seen in holo CARM1 structures (95). Recent structure-based optimization of this inhibitor class has


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resulted in a potent and selective probe compound suitable for in vivo experiments (97). The ability to achieve potent and selective inhibitors through different mechanisms of inhibition is exciting for the epigenetics field and provides needed tool compounds to elucidate the complex biology of these enzymes.

Structurally challenging PMTs: EZH2 and SETDB1 Despite marked success by structural biologists with many PMT families, some of the most interesting PMTs are not structurally enabled. As noted earlier, SETDB1, a member of the SUV39/EHMT subfamily, is expected to be a structurally distinct enzyme within the PKMT class. It contains a unique 347 amino acid sequence located within the SET domain, known as the bifurcation domain (98). Modeling of the position of the domain based on family member SETMAR indicates this domain may engage with the region of the protein involved in SAM binding (Boriack-Sjodin and Swinger, unpublished results), resulting in a SET domain architecture that could be unique among PMTs. Knowledge of the impact of the bifurcation domain on both the structure of the SET domain and changes to the structure of the SAM or peptide binding site may help to provide insight into this unique SET domain structure and its impact on enzyme activity and substrate specificity. EZH2 is a PKMT that is only active within a multiprotein complex. In vivo, EZH2 is found within the four component Polycomb Repressive Complex (PRC2; ~230 kD), although only three proteins, EZH2, SUZ12 and EED are required for catalytic activity (99). In 2012, a structure of PRC2 containing EZH2, Suz12, EED and RbAp48 was solved by electron microscopy and known structures of the individual components were placed into the reconstruction (100), but resolution limited a detailed analysis regarding the structure of EZH2.


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Crystal structures of truncated constructs of EZH2 have also been solved, however, the nucleotide active site conformations were incompatible with SAM binding and do not represent an active protein conformation (101, 102). Recent crystal structures of a minimal, catalytically active 170 kDa PRC2 with and without histone peptide bound from the thermohilic yeast Chaetomium thermophilum has provided the field with a detailed look at the extensive interactions between EZH2, SUZ12 and EED within the complex (103). The structure has also revealed the importance of SET activation loop (SAL) and its interactions with other EZH2 domains as well as SUZ12 and EED in maintaining the EZH2 SET domain in an active conformation. These interactions may explain the lack of activity from the truncated constructs. Analysis of the structure has provided potential insights into direct and allosteric regulation of EZH2 activity and has provided a scaffold on which to base future structural and mechanistic studies (103). Hypermethylation of the EZH2 substrate, H3K27, has been linked to cancer (104), making it a target for drug discovery efforts (105-109). Although potent, SAM-competitive inhibitors of EZH2 have entered the clinic, there are outstanding mechanistic questions which remain to be understood from a structural perspective. For example, EZH2 inhibitors show SAM competitive kinetic profiles but recent data showed generation of mutants outside the SET domain confer resistance to EZH2 inhibitors (110). The sequence of Chaetomium thermophilum EZH2 is quite divergent from that of human EZH2 with only 28% similarity and several sequence insertions and deletions (103), therefore it is not known whether the Chaetomium thermophilum complex can be used to investigate the mechanism of inhibition of these compounds. If the compounds are not cross-reactive with Chaetomium thermophilum PRC2, a structure of a potent EZH2 inhibitor bound to human or other mammalian EZH2 protein or the


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PRC2 complex would be required for elucidation of the inhibitory mechanism.. This example highlights the potential utility of structural biology at every stage of target validation.

Other methyltransferase enzymes Thus far, only enzymes that transfer methyl groups to protein residues have been discussed in detail. However, structural biology efforts are also active for other methyltransferase enzymes. DNA methyltransferases (DNMTs) are a small family of enzymes that methylate CpG islands. Hypermethylation of these groups has been shown to reduce tumor suppression (111) and nucleoside inhibitors that target DNMTs, azacitidine and decitabine, are in clinical use. Methyltransferase domains of four of the five DNMTs are known (6), including complexes with DNA (112, 113) and regulatory enzymes (114), providing a wealth of structural data for this family. In contrast, RNA methyltransferases belong to a much larger enzyme family with 57 members for which less than a third have been structurally characterized (6). Therefore, RNA methyltransferases provide new opportunities and challenges for structural biology researchers. Additional methyltransferase enzymes methylate small molecules including catechol and nicotinamide; structural biology efforts have been fruitful for these enzymes as well. For example, more than 40 structures of catechol-O-methyltransferase are available in the public domain. These have provided valuable insights into the catalytic mechanisms of these enzymes and have contributed to the design of potent and selective inhibitors (115).

Summary Structural biology has had tremendous impact on the study of protein methyltransferases. It has highlighted the similarity and diversity of the PMT enzyme families, provided insights on


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enzyme mechanism and substrate specificity, and guided efforts towards the design of selective and potent tool compounds to probe biology in normal cells and disease models. These efforts will continue, particularly for methyltransferases implicated in human disease. Going forward, structural characterization of methyltransferase enzymes with external domains beyond the catalytic domain, or in multiprotein complexes with and without substrates bound, will provide even more insight into the role of these additional domains on substrate recognition, binding, and regulation. Structural studies of larger proteins and multiprotein complexes will also increase our understanding of the environment of these catalytic domains in cellular settings. These larger complexes will likely be challenging and may require engagement of multiple structural methods in concert. Once coupled with information from biochemistry and cell biology, these more complicated structural studies will provide information critical to understanding these distinct, diverse and dynamic enzymes and their impact on human biology.

Acknowledgements The authors wish to thank R.A. Copeland, S. Ribich, A. Finley and W. Janzen for their critical reading of the manuscript and colleagues at Epizyme for helpful discussions.

Funding Sources All efforts were funded by Epizyme.

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Figure Legends Figure 1: Phylogenic trees of A) PKMT and B) PRMT enzymes based on chemogenic analysis (5). Proteins included in the PRMT tree are sequences with methyltransferase domains and include all PRMT enzymes as well as known RNA methyltransferases. Individual enzymes and protein families that are discussed in this review are highlighted with an asterisk.

Figure 2: Representative structures of protein methyltransferase enzymes. SAM and SAH are depicted as sticks in all panels. A. Ribbon depiction of PKMT SETD7 bound to SAH (1MT6 (116)). Structural features highlighted include the I-SET domain (yellow). B. Ribbon depiction of DOT1L (3QOW (5)). Structural features include the N-terminal domain (yellow) and the Cterminal α/β domain that binds SAM (green). C. Ribbon depiction of dimeric PRMT3 bound to SAH (1F3L (85)). Structural features include the methyltransferase domain (green), β-barrel domain (yellow), and dimerization domain (cyan).

Figure 3: Mechanism of A) PKMT and B) PRMT enzymes.

Figure 4: The auto-inhibitory loop of NSD family and SETD2 proteins can adopt multiple conformations. For all panels, the auto-inhibitory loop is cyan and the SET domain is green. A. Cartoon representation of NSD1 (3OOI (46)) bound to SAH in magenta sticks. The autoinhibitory post-SET loop is blocking the substrate pocket within the SET domain. B. WHSC1L1 (4YZ8, Temple et. al., 2015) bound to SAM has a disordered auto-inhibitory loop. C. SETD2 (4FMU (47)) bound to a propyl-substituted sinefungin analog depicted as orange sticks. The auto-inhibitory loop is flipped open such that the substrate pocket is more open. 30 ACS Paragon Plus Environment

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Figure 5: Cavernous binding sites in SMYD family enzymes allow diversity in ligand design. AC. Surface representations of SMYD1 (A; 3N71 (51)), SMYD2 (B; 3TG5 (53)) and SMYD3 (C; 5CCM (63)) are shown with N-terminal domain in light orange and C-terminal domain in light pink. Large cavernous binding pockets are formed at the domain interfaces. D. Protein-based overlays of ligands bound to SMYD family enzymes. p53 peptide and SAH from a SMYD2 structure (3TG5) are shown in cyan sticks. A-893 and SAM bound to SMYD2 (green; 4YND (44)), overlap with one trajectory of the peptide. EPZ030456 and SAM from a SMYD3 structure (5CCM; yellow) overlap with a different portion of the peptide.

Figure 6: PRDMs are structurally related to SET domains. A, PRMD2 (green, 2QPW (4)) is overlayed onto SETD7 (yellow, 1MT6 (116)). SAM is shown in magenta sticks. B. Human apo PRDM9 (yellow, 4IJD, Dong et al., 2012) is overlayed with murine PRMD9 (green, 4C1Q (71)) bound to peptide substrate (blue sticks) and SAM (magenta sticks). The auto-inhibitory loop that moves upon substrate binding is highlighted.

Figure 7: DOT1L is a dynamic protein methyltransferase. A. Surface representation of DOT1LSAM complex (3QOW (5)) is shown. N- and C-terminal domains are colored light orange and pink, respectively; SAM is shown in magenta stick representation. Flexible loops are depicted as ribbons; the activation loop is yellow. B. Surface representation of DOT1L-iodotubercidin complex (3UWP, Yu et. al., 2011) with same color scheme as A. Iodotubercidin is shown in green stick representation. Dramatic rearrangement of the activation loop is observed in the presence of different ligands. C. Cavity view of DOT1L (grey) bound to SAM (magenta;


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3QOW). D. Cavity view of DOT1L (grey) bound to pinometostat (cyan; 4HRA (79)). Ligand induced side chain rearrangements open up a larger cavity in the protein’s interior when the inhibitor is bound.

Figure 8: Diversity is found within PRMT family. Color scheme used in Figure 3C is reproduced for all panels. Nucleotides are represented as magenta spheres and ligands are represented by blue spheres. A. CARM1 bound to peptide and SAH (5DX0 (18)). B. CARM1 bound to an inhibitor and SAM (2Y1W (93)). C PRMT5 bound to EPZ015666 and SAM (4X61 (94)); MEP50 is in orange ribbon. D. PRMT6 bound to EPZ024011 and SAM (4Y30 (91)). E. PRMT3 bound to an allosteric inhibitor, SGC707 (4RYL (97)). In CARM1, PRMT5, and PRMT6, inhibitors bind in the same region as the peptide binds in CARM1. In contrast, the allosteric compound bound to PRMT3 resides in a distal location relative to the other inhibitors.


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Figure 1: Phylogenic trees of A) PKMT and B) PRMT enzymes based on chemogenic analysis (5). Proteins included in the PRMT tree are sequences with methyltransferase domains and include all PRMT enzymes as well as known RNA methyltransferases. Individual enzymes and protein families that are discussed in this review are highlighted with an asterisk. 368x165mm (300 x 300 DPI)

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Figure 2: Representative structures of protein methyltransferase enzymes. SAM and SAH are depicted as sticks in all panels. A. Ribbon depiction of PKMT SET7/9 bound to SAH (1MT6 (116)). Structural features highlighted include the I-SET domain (yellow). B. Ribbon depiction of DOT1L (3QOW (5)). Structural features include the N-terminal domain (yellow) and the C-terminal α/β domain that binds SAM (green). C. Ribbon depiction of dimeric PRMT3 bound to SAH (1F3L (85)). Structural features include the methyltransferase domain (green), β-barrel domain (yellow), and dimerization domain (cyan). 254x83mm (299 x 299 DPI)


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Figure 3:

Mechanism of A) PKMT and B) PRMT enzymes. 177x215mm (300 x 300 DPI)


Biochemistry

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Figure 4: The auto-inhibitory loop of NSD family and SETD2 proteins can adopt multiple conformations. For all panels, the auto-inhibitory loop is cyan and the SET domain is green. A. Cartoon representation of NSD1 (3OOI (46)) bound to SAH in magenta sticks. The auto-inhibitory post-SET loop is blocking the substrate pocket within the SET domain. B. WHSC1L1 (4YZ8, Temple et. al., 2015) bound to SAM has a disordered auto-inhibitory loop. C. SETD2 (4FMU (47)) bound to a propyl-substituted sinefungin analog depicted as orange sticks. The auto-inhibitory loop is flipped open such that the substrate pocket is more open. 323x83mm (299 x 299 DPI)


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Biochemistry

Figure 5: Cavernous binding sites in SMYD family enzymes allow diversity in ligand design. A-C. Surface representations of SMYD1 (A; 3N71 (51)), SMYD2 (B; 3TG5 (53)) and SMYD3 (C; 5CCM (63)) are shown with N-terminal domain in light orange and C-terminal domain in light pink. Large cavernous binding pockets are formed at the domain interfaces. D. Protein-based overlays of ligands bound to SMYD family enzymes. p53 peptide and SAH from a SMYD2 structure (3TG5) are shown in cyan sticks. A-893 and SAM bound to SMYD2 (green; 4YND (44)), overlap with one trajectory of the peptide. EPZ030456 and SAM from a SMYD3 structure (5CCM; yellow) overlap with a different portion of the peptide. 165x165mm (299 x 299 DPI)


Biochemistry

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Figure 6: PRDMs are structurally related to SET domains. A, PRMD2 (green, 2QPW (4)) is overlayed onto SETD7 (yellow, 1MT6 (116)). SAM is shown in magenta sticks. B. Human apo PRDM9 (yellow, 4IJD, Dong et al., 2012) is overlayed with murine PRMD9 (green, 4C1Q (71)) bound to peptide substrate (blue sticks) and SAM (magenta sticks). The auto-inhibitory loop that moves upon substrate binding is highlighted. 127x77mm (299 x 299 DPI)


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Biochemistry

Figure 7: DOT1L is a dynamic protein methyltransferase. A. Surface representation of DOT1L-SAM complex (3QOW (5)) is shown. N- and C-terminal domains are colored light orange and pink, respectively; SAM is shown in magenta stick representation. Flexible loops are depicted as ribbons; the activation loop is yellow. B. Surface representation of DOT1L-iodotubercidin complex (3UWP, Yu et. al., 2011) with same color scheme as A. Iodotubercidin is shown in green stick representation. Dramatic rearrangement of the activation loop is observed in the presence of different ligands. C. Cavity view of DOT1L (grey) bound to SAM (magenta; 3QOW). D. Cavity view of DOT1L (grey) bound to pinometostat (cyan; 4HRA (79)). Ligand induced side chain rearrangements open up a larger cavity in the protein’s interior when the inhibitor is bound. 229x142mm (299 x 299 DPI)


Biochemistry

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For Table of Contents use only 125x49mm (300 x 300 DPI)


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Protein Methyltransferases: A Distinct, Diverse, and Dynamic Family of

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