Characterization of SETD3 methyltransferase – mediated protein methionine methylation

Most characterized protein methylation events encompass arginine and lysine N -methylation, and only a few cases of protein methionine thiomethylation have been reported. Newly discovered oncohistone mutations include lysine-to-methio-nine substitutions at positions 27 and 36 of histone H3.3. In these instances, the methionine substitution localizes to the active-site pocket of the corresponding histone lysine methyltransferase, thereby inhibiting the respective transmethylation activity. SET domain – containing 3 (SETD3) is a protein ( i.e. actin) histidine methyltransferase. Here, we generated an actin variant in which the histidine target of SETD3 was substituted with methionine. As for previously characterized histone SET domain proteins, the methionine substitution substantially (76-fold) increased binding affinity for SETD3 and inhibited SETD3 activity on histidine. Unexpectedly, SETD3 was active on the substituted methionine, generating S -methylmethionine in the context of actin peptide. The ternary structure of SETD3 in complex with the methionine-containing actin peptide at 1.9 Å resolution revealed that the hydrophobic thioether side chain is packed by the aromatic rings of Tyr 312 and Trp 273 , as well as the hydrocarbon side chain of Ile 310 . Our results suggest that plac-ing methionine properly in the active site — within close proximity to and in line with the incoming methyl group of SAM — would allow some SET domain proteins to selectively methylate methionine in proteins. S -methylmethionine ( SMM ), which itself serves as a methyl donor for homocysteine ( Hcy ) methylation, generating two molecules of methionine. Homocysteine is a nonproteinogenic amino acid, differing from methionine and cysteine by lacking a terminal methyl group (CH 3 ) or an additional methylene bridge (CH 2 ), respectively. Like cysteine, homocysteine carries a reac- tive thiol nucleophile. In humans, the level of homocysteine, an increaseof which is considered a risk factor for cardiovascular diseases, is regulated by at least three pathwaysto regenerate methionine:(i)methionine synthase; (ii) betainehomocysteine S -methyltransferase(BHMT-1) (56), using the methyldonorbeta- ine (also known as trimethylglycine); and (iii) betaine-homocysteine S -methyltransferase 2 (BHMT-2), which uses S -methylmethionine, but not betaine, as a methyldonor(57). Inaddition,a pathogenic bacterial secreted effectorprotein NleE methylates cysteine- S ina humanzinc fingerprotein.

Post-translational methylation on proteins, particularly histones, plays a fundamental role in regulating gene expression and chromatin organization (1)(2)(3). For example, histone H3 methylation at lysine residues 4,9,27,36, and 79 signals for gene activation or repression of transcription. Dysregulation of these epigenetic signals has been correlated with and mechanistically linked to many human disorders including cancer (4)(5)(6). Recurrent mutations in histone H3.3, first discovered in pediatric high-grade glioma and since found in multiple other cancer types, have been found to lead to alterations in the epigenetic landscape that are associated with the pathology of these diseases (7,8) (reviewed in Ref. 9 and the references therein). The most heavily characterized oncohistone mutations are the ly-sine-to-methionine mutants at K27M and K36M, which perturb the methylation site for specific lysine methyltransferases (MTases) and dominantly inhibit methylation of corresponding lysine residues in other histone H3 proteins. Structural characterization of histone MTases in complex with the methioninesubstituted histone H3 peptides, including G9a-H3K9M (10,11), PRC2-H3K27M (12), and SETD2-H3K36M (13), revealed that the methionine residue localizes to the active-site pocket that normally accommodates the target lysine residue, thereby inhibiting the respective transmethylation activity. In vitro, it was the study of LSD1 (a histone H3K4me2/1 demethylase) that first used a H3 peptide bearing methionine in place of methyl-Lys 4 (PDB code 2V1D), which showed that the K4M peptide is a strong inhibitor of LSD1 because the methionine substitution led to a 30-fold increase in binding affinity (14). Interestingly, EZHIP (enhancer of zeste homologs inhibitory protein) inhibits PRC2 methylation activity by presenting a methionine-containing, naturally occurring, H3K27M-like sequence (15)(16)(17)(18), suggesting a broader role for these sequences in normal regulatory processes.
Biochemically, the methyl donor S-adenosyl-L-methionine (SAM) is the ATP-activated form of methionine (19), which contains a reactive positively charged methylsulfonium group (CH 3 -S 1 ) ( Fig. 1) (56,57). In flowering plants, the biosynthesized L-methionine can be methylated to S-methylmethionine by a SAM-dependent reaction, catalyzed by methionine S-MTase (20). Although the biochemistry of methionine methylation is best characterized in the context of small molecules, the significance of protein methionine methylation is less clear. Reports of protein methionine methylation are limited to two examples in single-cell organisms involving a partially purified enzyme activity active on cytochrome c in the flagellate Euglena gracilis (21) and an automethylation of LaeA in the filamentous fungus Aspergillus nidulans (22).
SETD3 was identified as the first metazoan histidine MTase that works on an actin histidine residue (23)(24)(25)(26). We have noted that SETD3 shares a high degree of global similarity with two characterized protein lysine MTases (human SETD6 and plant rubisco LSMT) and detected that SETD3 has weak lysine methylation activity on an actin variant in which the target His 73 is substituted by a lysine (H73K) (27,28). Prompted by the inhibitory effect of oncohistone mutations of lysine-to-methionine and similar endogenous MTases inhibiting sequences, here we report that unexpectedly, SETD3 has methylation activity on an actin variant in which the target His 73 is substituted by a methionine (H73M). We investigate the structural and molecular determinants of generating S-methylmethionine in the context of a protein peptide.

SETD3 methylation of methionine
Our previous characterizations of SETD3 indicated that SETD3 exhibited in vitro activity on full-length recombinant actin-Lys 73 , albeit much weaker than actin-His 73 activity (27) (Fig. 2A). Unexpectedly we observed SETD3 activity on actin-Met 73 , in which the target His 73 was substituted by a methionine ( Fig. 2A). As a negative control, no activity was observed on actin-Ala 73 , in which His 73 was replaced by an alanine. Consistent with the full-length recombinant protein, SETD3 exhibited activities on peptide substrates bearing either histidine, lysine, or methionine at position 73 (Fig. 2B). Under the saturating conditions of overnight reactions in which SETD3 completed reactions on its native substrate His 73 , we observed ;50% reduced activity on Met 73 and much reduced activity on Lys 73 .
As aforementioned, the methyl donor SAM carries a positively charged methylsulfonium group (CH 3 -S 1 ), and SAMdependent MTases require the target atom be in a deprotonated state. In agreement with this mechanism, SETD3 has varied, substrate-dependent optimum pH values: an optimum pH of 7 and above for histidine where the imidazole ring (with a typical pK a value near 6) is uncharged and a pH of ;10.5 for lysine (mainly because of its typical pK a value of ;10) (27). In comparison, methionine has a nonpolar S-methyl thioether side chain, and SETD3 has a relatively constant activity on Met 73 over a pH range of 5-11 with a small increase of activity at pH 8.0 (Fig.  2C) at the linear time point of 1 h (Fig. 2D). Although SETD3 has a catalytic efficiency k cat /K m of 1.8 h 21 mM 21 on His 73 methylation (28), SETD3 is a slow enzyme on Met 73 without turnover (i.e. the product over the enzyme concentration is less than 1; [SAH]/ [E] , 1). Under the first-order kinetics conditions, we estimated that SETD3 showed an activity on Met 73 with an apparent k cat / K m value of ;0.12 h 21 mM 21 (Fig. 2E).
Next, we measured the dissociation constant between SETD3 and Met 73 peptide by isothermal titration calorimetry (Fig. 2F). We observed a K D value of 25 nM, an increase of binding by a factor of 76 from the SETD3 dissociation of His 73 peptide, which might partially explain the low turnover rate of Met 73 from SETD3. This result of enhanced binding (reduced K D value) by the methionine-substituted peptide agrees with previous studies on LSD1 (;30-fold) (14) and PRC2 (;20-fold) (12). In addition, the 25 nM dissociation constant matches well with the K D value of 32 nM observed between G9a and H3 peptide with the Lys 9 replacement with norleucine in the presence of added SAM (11). Under the conditions that activity of SETD3 on Met 73 is negligible (Fig. S1A), the Met 73 peptide inhibits SETD3 activity on His 73 with an IC 50 of 1 mM (Fig. 2G), consistent with G9a inhibition by H3 peptide K9M with IC 50 of 2.3 mM (11).
Structure of SETD3 in complex with Met 73 -containing peptide SETD3 was readily crystallized with the Met 73 (residues 66-88) peptide in the presence of SAH, and we observed electron In plants, free amino acid methionine (Met) is methylated to become S-methylmethionine (SMM), which itself serves as a methyl donor for homocysteine (Hcy) methylation, generating two molecules of methionine. Homocysteine is a nonproteinogenic amino acid, differing from methionine and cysteine by lacking a terminal methyl group (CH 3 ) or an additional methylene bridge (CH 2 ), respectively. Like cysteine, homocysteine carries a reactive thiol nucleophile. In humans, the level of homocysteine, an increase of which is considered a risk factor for cardiovascular diseases, is regulated by at least three pathways to regenerate methionine: (i) methionine synthase; (ii) betaine homocysteine S-methyltransferase (BHMT-1) (56), using the methyl donor betaine (also known as trimethylglycine); and (iii) betaine-homocysteine S-methyltransferase 2 (BHMT-2), which uses S-methylmethionine, but not betaine, as a methyl donor (57). In addition, a pathogenic bacterial secreted effector protein NleE methylates cysteine-S in a human zinc finger protein.
density for peptide residues 66-84 (Fig. 3A). The ternary structure was determined to resolution of 1.9 Å (Table S1). The SETD3-Met 73 structure is virtually identical to the previously determined structures of SETD3 in complex with His 73 (PDB code 6MBJ), methylated His 73 (PDB code 6OX2), or Lys 73 (PDB code 6OX3) with pairwise comparison of less than 0.2 Å of root-mean-square deviation across two complexes of each crystal structure in the crystallographic asymmetric unit in space group P2 1 (Fig. 3B). In the methionine-occupied active site ( Fig.  3C), the sulfur atom (Sd) of Met 73 is 5.3 Å away from the sulfur atom of SAH, in which a transferable methyl group would be attached (Fig. 3D). The observed conformations of the Met 73 side chain and the methionine moiety of SAH in the active site is nearly symmetric to each other by a rotation of 180°. The nonpolar S-methyl thioether side chain is packed in between the aromatic rings of Tyr 312 (via the Cg atom) and Trp 273 . The terminal Ce carbon of Met 73 is in van der Waals contact with the main chain Ca atom of Asp 274 (3.4 Å), the indole ring of Trp 273 (3.5 Å), and the side chain of Ile 310 (4.0 Å) (Fig. 3E). In addition, the main chain Ca carbon of Met 73 of actin forms a C-H···O type of hydrogen bond (29) with side chain of Asn 255 (Fig. 3F). The dominant nonpolar interactions give rise to favorable binding of Met 73 in an active site constructed mainly of aromatic and hydrophobic residues; a greasy environment helps to deprotonate (lower the pK a value) lysine, as evident by the observed pK a value of 6.5 for a lysine fully buried inside the hydrophobic core in a mutant T4 lysozyme (30). Thus, the mutations of the polar residue Asn 255 required for hydrogen bonding with His 73 during catalysis (27) to hydrophobic residues alanine and valine improved activity for Met 73 methylation by ;3-fold (Fig. 3, G and H).
Next, we determined the structure of mutant N255V in complex with the Met 73 peptide to resolution of 1.76 Å (Table S1). The mutant structure is nearly identical to that of the WT enzyme, with a root-mean-square deviation of 0.16 Å across 946 pairs of Ca atoms (Fig. 3, I-J). The increased hydrophobicity of Val 255 , accompanied with the loss of polar interaction associated with Asn 255 , allows the side chain of Met 73 of actin to move slightly toward to the methyl donor (Fig. 3I).

Comparison of SETD3 with other MTases in binding of methionine
We compared the structures of the SETD3-Met 73 complex with that of three histone H3 MTases (G9a, PRC2, and SETD2) in complex with peptides containing methionine mutations at lysine 9, 27, and 36, respectively (10,12,13). By superimposing the respective cofactor SAM or SAH, we observed two major differences. First, the histone H3 peptides and actin peptide run in opposite directions (Fig. 4A). Second, the methionine residues in the context of histone H3 are further away from the transferrable methyl group of SAM, whereas in the actin peptide, the methyl acceptor (sulfur Sd) is located within a shorter distance of and in line with the incoming methyl group of the Protein methionine methylation methylsulfonium of SAM (Fig. 4B). Such a linear arrangement comprising the nucleophile (Met-S:), the methyl group, and the leaving thioester group of SAM in the transition state is required for the S N 2 reaction mechanism used by SAM-dependent MTases (Fig. 4C).
Next, we tested three additional SET domain proteins, SET7 (31), PR/SET domain 9 (PRDM9) (32), and G9a-like protein (GLP) (33), on histone peptides containing methionine substitution at either lysine position 4 or 9. Each enzyme is active on its respective lysine-containing peptide substrate (Fig. 4D) but not the peptides with the corresponding methionine substitution. The activity observed with PRDM9 on H3M4 peptide is probably due to the automethylation of PRDM9 (Fig. 4E), because intramolecular automethylation was observed previously on lysine residues by PRDM9 (34) (Fig. S2).
The difference of activity on methionine by SETD3 as compared with other SET domain proteins on histone H3 probably reflects the difference of their natural substrates: the smaller histidine residue needs to enter deeper into the active site of SETD3 and positions the target nitrogen atom pointing toward, and within a short distance of, the incoming methyl group, whereas the terminal nitrogen atom of the larger lysine residue in an extended side chain conformation can be positioned in the same target position even with the backbone of peptide being further away (Fig. 4F). The inactivity on methionine by histone MTases presumably reflects the fact that the methionine side chain is not juxtaposed in close enough proximity to the donor methyl group of SAM for nucleophilic attack (Fig.  4B). We note that in the case of SETD3 bound with Lys 73 peptide, the side chain of Lys 73 is in a bent conformation (Fig. 3B) (27,28). We also note that, unlike G9a (11), we did observe stable interactions between GLP and histone H3K9M in the presence of either SAM or SAH (Fig. S3), which forms heterodimer with G9a (33). However, the affinity for GLP-H3K9M-SAM (K D = 120 nM) is less than four times weaker than that of G9a.

SETD3 generates S-methylmethionine
HPLC-MS/MS confirmed and localized the methylation of actin peptide on Met 73 by SETD3 resulting in S-methylmethionine. For the unmodified peptide, the only observable signal is from the expected mass of the peptide (2855.378 Da, 0.35 ppm error) and oxidized forms (Fig. 5A). A major precursor with a mass shift consistent with methylation (2869.390 Da, 0.94 ppm error) was identified when actin was reacted with SETD3 (Fig.  5B). The 14 charge state precursor of the modified peptide was isolated and fragmented by method of electron transfer dissociation (ETD).
ETD of the modified peptide resulted in an unambiguous localization of a 114.012 Da (which equals 2869.390-2855.378) mass shift to Met 73 (Fig. 5C). This evidence suggests two possible additions. The first is that CH 2 is added and results in a mass change of 114.016 Da (calculated). The second possibility is that the addition of a CH 3 to the sulfur results in an addition of 115.0235 Da (calculated), and the formation of a positively charged sulfonium ion that results from the loss of an electron, when the valence shell is expanded to accommodate the additional bond; however, this fixed positive charge would normally be accounted for by a proton, which therefore must be subtracted from the uncharged mass, also resulting in a final mass difference of 14.016 Da (which equals 15.0235-1.0079). Thus, the mass of the modification alone does not distinguish between these two modes of methylation.
Changes in retention time support the assignment of Smethylmethionine (Fig. 5D). The methyl modified peptide elutes 3 min earlier than the unmodified peak on our reversed phase C3 column. Such a shift is consistent with a decrease in hydrophobicity, consistent with the addition of a strongly hydrophilic fixed positive charge. Monomethylation of carbon, which seems unlikely, increases hydrophobicity and would result in the opposite: a shift to longer retention times. Thus, the shift in retention time is consistent with the S-methylmethionine and the formation of a fixed positive charge and highly hydrophilic sulfonium.
In addition, we also note unusual charge state distributions and mass-to-charge ratios in the ETD-based fragmentation that could be explained by the unusual peptide chemistry from the fixed charged sulfonium ion (Fig. 5E). Usually during fragmentation, fragment ions are almost exclusively observed in an m/z range of 400-1200 because of correlation between the mass and the number of basic sites. Here, we observed 11 fragment ions extending to 2000 m/z. This unusual distribution of ions is likely due to the fixed positive charge of the S-methylmethionine.
There are multiple lines of evidence for assigning the product as S-methylmethionine based on intact mass, fragment sequence coverage, chromatographic shifts toward more hydrophilic retention times, and unusual behavior in fragmentation. The precedent for this type of reaction and sulfonium ions in general is readily apparent in the production of SAM, which is itself a sulfonium ion carrying a fixed positive charge. Thus, the proposed reaction that produces S-methylmethionine would start and end with a sulfonium ion (Fig. 4C).
Likely because of the unique chemical nature of the sulfonium moiety of the S-methylmethionine, we did not observe fragments that include modified Met 73 , using collision-induced dissociation (CID) or high-energy collision dissociation (HCD). It is not clear why the proposed sulfonium structure significantly changes the characteristic of the peptide. However, it is our conjecture that it interferes with the mobile proton associated with HCD/CID dissociation method (35).

Discussion
A wide variety of SAM-dependent protein methylation occurs on nitrogen atoms of the protein N terminus and the side chains of arginine, lysine, glutamine, and histidine and the carboxylate oxygen atoms of the side chains of glutamate and aspartate, as well as the protein C terminus (36). In addition to two previous reports (21,22), we show here that the sulfur of methionine, in the context of an actin variant, can be methylated by the SET-domain MTase SETD3 in vitro. We note that previous examples of protein methionine methylation are generated by either an unidentified methyltransferase in E. gracilis (21) or LaeA of A. nidulans, a member of the seven-strand MTase family (22) that is structurally distinct from the SETfamily MTases (37).
Identification and localization of methylation on methionine required use of ETD, a technique that is infrequently used in bottom-up proteomics. Thus, it is unlikely that previous Protein methionine methylation proteomic analyses would have localized methylation to methionine residues. Future whole proteome profiling with ETD targeting methionine methylation may yield a host of novel modifications and better explain previously obfuscated mechanisms. Further, by including methionine methylation as a potential post-translational modification, one may prevent incorrect assignments. The unusual fragmentation characteristics observed may be due to the nature of the peptide, and thus further efforts are required for assigning a pattern for methionine methylation.
In addition to methionine-S-methylation, protein cysteine-S-methylation has been reported in a zinc-coordinating cysteine in a human zinc finger protein, methylation of which is catalyzed by a pathogenic bacterial secreted effector protein NleE and results in loss of zinc binding (38). The Escherichia coli DNA repair protein Ada automethylates by transferring the aberrant methyl group from damaged DNA (methyl phosphotriester lesion or mutagenic O 6 -methylguanine lesion) to one of its own cysteine residues (39). Methionine and cysteine are the two common sulfur-containing amino acids, the former being hydrophobic and the latter being highly nucleophilic. Thiomethylation switches these properties of the two amino acids ( Fig. 1): S-methylmethionine contains a highly reactive and charged methylsulfonium group, whereas S-methylcysteine mostly results in elimination of cysteine's intended function (39)(40)(41)(42). The charged methylmethionine had an effect on the pI of cytochrome c from E. gracilis (21). We note that a human S-methyltransferase (Thiopurine methyltransferase) discovered several decades ago (43,44) that is an important drug-metabolizing enzyme active on thiopurine drugs (45,46) has no known natural substrates even now. Two human enzymes have been reported to have automethylation on cysteine residues (42,47).
The hydrophobicity of methionine within substrate peptides is well-accommodated in aromatic-rich active sites, and methionine replacement of the target His 73 leads to an impressive increase in binding affinity for SETD3, as well as for the peptides with methionine substitution of target lysine of histone modifying enzymes (LSD1 and PRC2), effectively inhibiting the enzymatic activities on their native substrates. We show that placement of methionine properly in the active site-close distance to and in line with the incoming methyl group (Fig. 4B)allows SETD3 and its mutant variants to methylate methionine. In humans, many of the 55 SET domain-containing proteins (48) have not been fully characterized, and further study could potentially reveal that some of them can selectively methylate methionine in a protein context.

Crystallography
A ternary complex of SETD3, Met 73 peptide (residues 66-88), and SAH was formed by mixing the three components in a molar ratio of 1:4:5 (protein:Met 73 :SAH) and incubated on ice for 1 h. For the ternary complex of SETD3 mutant N255V, Met 73 (residues 66-88) peptide, and SAM, the components were mixed at a molar ratio of 1:5:10 (protein:Met 73 :SAM) and incubated on ice overnight. Sitting drops (0.4-ml) at ;20°C were set up by an Art Robbins Gryphon Crystallization Robot containing the ternary complexes (;14 mg/ml or ;0.2 mM) plus the well solutions of 0.2 M ammonium acetate, 0.1 M sodium citrate tribasic dihydrate, pH 5.6, and 30% (w/v) PEG 4000.
Cryoprotectant buffer containing 25% (v/v) ethylene glycol and the crystallization solution was used to flash-freeze single crystals. The SER-CAT Beamline 22ID of the Advanced Photon Source at Argonne National Laboratory was used remotely to collect X-ray diffraction data, processed with HKL2000 (49). The previously solved structure of human SETD3 (PDB code 6MBJ) was used as the search model in the molecular replacement of PHENIX PHASER module (50,51). Randomly chosen 5% reflections were used for the validation of the R free value throughout the process of structure refinement with PHENIX REFINE (52). Manual building and corrections of structural models between refinement rounds were carried out using COOT (53,54). The PDB validation server was used to ratify the quality of the final refined structure. The molecular graphic program PyMOL (Schrödinger, LLC) was used to generate figure panels.

Methylation assay
Methylation assays on full-length actin were performed as previously described (27). Briefly, batch-purified GST-SETD3 (2 mM) and GST-b-actin (1 mM) were reacted with 2 mCi of For peptide substrates, the Promega bioluminescence assay (MTase-Glo TM ) (55) was used to measure the methylation reaction by-product SAH, which is converted into ATP in a two-step reaction, and ATP is detected through a luciferase reaction. A low-volume 384-well plate with each well containing an aliquot of 5 ml of reaction mixture was used to measure luminescence signal by a Synergy 4 multimode microplate reader (BioTek).

Optimal pH analysis for actin M73 peptide
A reaction mixture contained 3.6 mM SETD3, 50 mM Met 73 peptide, 40 mM SAM, 50 mM NaCl, 0.1 mg/ml BSA, 1 mM DTT, and varied pH with 20 mM citric acid, bis-tris propane (below pH 9) or 20 mM glycine/NaOH (above pH 9). The 20 mM citric acid, bis-tris propane is a mixture of 10 mM citric acid and 10 mM Bis-Tris propane. The reaction was performed at 37°C for 1 h, terminated by the addition of TFA to a final concentration of 0.4% (v/v), and then diluted 4-fold with 20 mM Tris-HCl, pH 8.0, 50 mM NaCl, 0.1 mg/ml BSA, and 1 mM DTT (the reaction buffer).

Time-course measurement
The reaction mixture contained 0.36 mM enzyme (SETD3, N255A, and N255V), 20 mM Met 73 peptide (residues 66-88), and 40 mM SAM in the reaction buffer. The reaction was performed at 37°C and was terminated at the indicated time with the addition of 0.1% (v/v) TFA to 10-ml reaction aliquot.

Single-turnover kinetics
The reaction mixture of 20 ml contained SETD3 ([WT] = 1.44 mM, [N255A/V] = 0.72 mM), 40 mM SAM, and varying concentration of Met 73 peptide in the reaction buffer. The reactions were carried out at 37°C for 1 h. The apparent k cat /K m value was estimated from the slope of low substrate concentration data point where the reaction rate varies linearly with substrate concentration.

Inhibition of SETD3 activity on His 73 by Met 73
A reaction mixture contained 0.18 mM SETD3, 40 mM SAM, and incubated with increasing concentration of Met 73 peptide (0-100 mM) in the reaction buffer for 30 min. The addition of 20 mM His 73 peptide started the reaction for 20 min at room temperature.

Isothermal titration calorimetry
A MicroCal PEAQ-ITC automated system (Malvern Instrument Ltd.) was operated at 25°C. SETD3 (20 mM in 20 mM Tris-HCl, pH 8.0, 50 mM NaCl, 5% glycerol, and 0.5 mM tris (2-carboxyethyl) phosphine) was maintained in the sample cell. The actin peptides (400 mM) in the identical buffer were injected into the cell by a syringe under continuous stirring (750 rpm) with the reference power set as 8 mcal/s. The volume of each injection was 2 ml with a fixed duration time of 4 s, and the spacing time between the injections was 250 s to achieve equilibrium. Binding constants were calculated by fitting the data using a single-binding site model by the ITC data analysis program supplied by the manufacturer. We also measured GLP (20 mM in the reaction buffer) binding to histone H3 peptide Met 9 (residues 1-21) (200 mM) in the presence of SAH or SAM (50 mM).

HPLC-MS/MS
Actin peptides were desalted, dried, and resuspended with 2% acetonitrile and 0.1% formic acid, resulting in a final concentration of 10 mM. HPLC was performed on a Dionex U-3000 Pro-flow system. A 20-min gradient was performed from 1 to 35% of 98% acetonitrile and 0.1% formic acid. An aliquot of sample (1 ml) was loaded onto a C-18 column with a 100-mmdiameter and 10-cm-long (Waters XBridge TM C18 3.5 mM 130 Å).
Acquisition was performed by a Thermo Scientific Orbitrap Fusion Lumos. A static spray voltage of 2000 V and an iontransfer tube temperature of 320°C were set for the source. MS1 was performed by the Orbitrap in positive mode, with the ETD parameters listed in Table S2.
Two m/z peaks were targeted for MS2 fragmentation: 714.8508 (for the unmodified actin peptide) and 718.3551 (for the modified actin peptide) both are 14 ions. Two different ETD parameters were used (Table S2), resulting in two different fragment spectra per duty cycle per precursor.
Multiple other MS settings were tested, most notably CID and HCD for MS2 fragmentation and MS3 using ETD and CID/HCD. These attempts did not yield sufficient coverage or interpretable data and are not presented. Raw files were initially analyzed by our in-house software and confirmed with Pro-Sight 4.0 (Thermo Scientific). Final analysis for presented spectra was performed manually, and all annotations were manually validated.

Data availability
The X-ray structures (coordinates and structure factor files) have been submitted to the PDB under accession numbers 6WK1 (SETD3-Met 73 -SAH) and 6WK2 (N225V-Met 73 -SAM).
The MS proteomics data have been submitted to the Proteo-meXchange via the PRIDE database (accession PXD019203).