SulfurâOxygen Chalcogen Bonding Mediates AdoMet Recognition in

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Sulfur - Oxygen Chalcogen Bonding Mediates AdoMet Recognition in the Lysine Methyltransferase SET7/9 Robert J. Fick, Grace M. Kroner, Binod Nepal, Roberta Magnani, Scott Horowitz, Robert L. Houtz, Steve Scheiner, and Raymond C. Trievel ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.5b00852 • Publication Date (Web): 29 Dec 2015 Downloaded from http://pubs.acs.org on January 4, 2016

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ACS Chemical Biology

Sulfur - Oxygen Chalcogen Bonding Mediates AdoMet Recognition in the Lysine Methyltransferase SET7/9

Robert J. Fick1, Grace M. Kroner1, Binod Nepal2, Roberta Magnani3, Scott Horowitz4,5, Robert L. Houtz3, Steve Scheiner2, and Raymond C. Trievel1* Departments of Biological Chemistry1 and Molecular, Cellular, & Developmental Biology4, University of Michigan, Ann Arbor, MI 48109, USA 2

Department of Chemistry and Biochemistry, Utah State University, Logan, UT 84322, USA

3

Department of Horticulture, University of Kentucky, Lexington, KY 40546, USA

5

Howard Hughes Medical Institute, Ann Arbor MI 48109 USA

*Corresponding Author: Raymond Trievel Address:

University of Michigan Department of Biological Chemistry 1150 West Medical Center Drive 5301 Medical Science Research Building III Ann Arbor, MI 48109

E-mail:

[email protected]

Phone:

734-647-0889

FAX:

734-763-4581

ACS Paragon Plus Environment


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ABSTRACT Recent studies have demonstrated that carbon-oxygen (CH•••O) hydrogen bonds have important

roles

in

S-adenosylmethionine

(AdoMet)

recognition

and

catalysis

in

methyltransferases. Here, we investigate noncovalent interactions that occur between the AdoMet sulfur cation and oxygen atoms in methyltransferase active sites. These interactions represent sulfur – oxygen (S•••O) chalcogen bonds in which the oxygen atom donates a lone pair of electrons to the σ anti-bonding orbital of the AdoMet sulfur atom. Structural, biochemical, and computational analyses of an asparagine mutation in the lysine methyltransferase SET7/9 that abolishes AdoMet S•••O chalcogen bonding reveal that this interaction enhances substrate binding affinity relative to the product S-adenosylhomocysteine. Corroborative quantum mechanical calculations demonstrate that sulfonium systems form strong S•••O chalcogen bonds relative to their neutral thioether counterparts. An inspection of high-resolution crystal structures reveals the presence of AdoMet S•••O chalcogen bonding in different classes of methyltransferases, illustrating that these interactions are not limited to SET domain methyltransferases. Together, these results demonstrate that S•••O chalcogen bonds contribute to AdoMet recognition and can enable methyltransferases to distinguish between substrate and product.

Graphical Abstract

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S-adenosylmethionine (AdoMet)-dependent methyltransferases have fundamental roles in numerous biological processes including metabolism, signal transduction, and gene regulation.1-3 These enzymes belong to multiple structurally distinct classes and utilize AdoMet as the methyl donor in catalyzing the methylation of a wide array of substrates, including amino acids, cofactors, lipids, neurotransmitters, DNA, RNA, and proteins. A recent survey of methyltransferase crystal structures has illustrated that oxygen atoms within the active sites of these enzymes form unconventional carbon-oxygen (CH•••O) hydrogen bonds with the sulfonium cation of AdoMet.4 Biochemical and biophysical studies of the lysine methyltransferase (KMT) SET7/9 demonstrated that these hydrogen bonds promote AdoMet binding and facilitate transition state stabilization for the SN2 methyl transfer reaction.4, 5 In the course of characterizing CH•••O hydrogen bonding in SET7/9, we observed a 3.0 Å interaction between the AdoMet sulfur atom and the carboxamide oxygen atom of Asn265 (Figure 1A). Structures of SET7/9 ternary complexes bound to AdoMet and small molecule inhibitors also display similar S•••O interaction distances (3.0 – 3.2 Å).6 As the minimum van der Waals contact distance for sulfur and oxygen is 3.3 Å, this interaction is too short to represent a van der Waals interaction and is indicative of an S•••O chalcogen bond between the AdoMet sulfonium cation and Asn265 carboxamide group. Chalcogen bonds occur when an atom donates a lone pair of electrons into the σ antibonding orbital of a chalcogen atom (O, S, Se, Te, and Po).7 These interactions are frequently categorized as σ-hole bonds that include the more widely appreciated halogen bonds, as well as tetrel and pnictogen bonds.8-13 Quantum mechanical (QM) calculations have demonstrated that the strengths of chalcogen bonds are comparable to weak hydrogen bonds, whereas interactions involving a chalcogen cation, such as the AdoMet sulfonium cation, can display interaction

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energies that are tenfold greater than their neutral counterparts.12, 13 With respect to biological macromolecules, S•••O, and S•••N, and S•••S chalcogen bonds involving methionine and cystine have been identified in proteins through surveys of crystal structures and have been proposed to promote protein folding and stability.14-18 Chalcogen bonding has also been observed in proteinligand complexes, and efforts are being undertaken to exploit these interactions in drug design.19, 20

However, the potential contributions of chalcogen bonds in enzyme catalysis and substrate

binding remain largely unexplored,18 particularly in the context of AdoMet-dependent methyltransferases.

RESULTS AND DISCUSSION Prompted by these observations, we sought to ascertain whether the S•••O chalcogen bond between the AdoMet sulfonium cation and the Asn265 carboxamide group in SET7/9 contributes to substrate recognition or catalysis. We chose to employ SET7/9 to characterize this interaction, as it has served as a well-characterized model for studying CH•••O hydrogen bonding to AdoMet,4, 5, 21 and the S•••O chalcogen bond can be readily probed by mutating Asn265 to alanine, thus abolishing the interaction. Prior to characterizing the effect of this substitution, we determined the structure of the catalytic domain of the SET7/9 N265A mutant bound to the products S-adenosylhomocysteine (AdoHcy) and a TAF10 K189me1 peptide at 1.55 Å resolution (Figure 1B and 1C, and Supplementary Table 1). Structural alignment of the SET7/9 N265A mutant and wild type (WT) enzyme illustrates that the structures are highly homologous, with a Cα RMSD of 0.19 Å for all aligned atoms. A comparison of the structures reveals that the N265A substitution does not perturb the conformation of the neighboring active site residues, nor do ordered water molecules occupy the space vacated by the Asn265 side chain

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in the SET7/9 N265A mutant (Figure 1). To further corroborate these observations, we examined the thermodynamic stability of the N265A mutant using differential scanning calorimetry (DSC) and measured a melting temperature (TM) of 44.3 °C (Supplementary Figure 1), which is modestly reduced compared to WT SET7/9 (TM = 46.6 °C). These results indicate the N265A substitution slightly destabilizes the enzyme, consistent with the loss of hydrogen bonding and van der Waals interactions with the Asn265 side chain (Figure 1A). Finally, we examined whether the N265A substitution alters protein substrate recognition by measuring the binding of a TAF10 peptide substrate to the mutant using isothermal titration calorimetry (ITC) and observed that its dissociation constant differs by less than two-fold compared to the WT enzyme (Table 1 and Supplementary Figure 2). After establishing that the N265A mutation does not alter the structure or protein substrate binding affinity of SET7/9, we characterized the AdoMet and AdoHcy dissociation constants and steady state kinetic parameters of the N265A mutant. The N265A substitution decreases the binding affinity for AdoMet by eight-fold, whereas the mutation diminishes the affinity for AdoHcy by only two-fold (Table 1). Correlatively, the KM value of AdoMet for the SET7/9 N265A mutant is increased by seven-fold compared to the WT enzyme, illustrating a corresponding loss in substrate recognition by the mutant. These results concur with QM calculations demonstrating that sulfonium cations form stronger S•••O chalcogen bonds compared to cognate neutral thioethers.13 Thus, the S•••O chalcogen bond to AdoMet permits SET7/9 to distinguish between the substrate and AdoHcy, thus mitigating product inhibition. In addition to the diminished affinity for AdoMet, the kcat value of the SET7/9 N265A mutant is diminished seven-fold in comparison to the WT enzyme. As methyl transfer has been shown to be at least partially rate limiting in SET7/9,4 the reduced kcat value implies a slight catalytic

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defect in the N265A mutant. It is conceivable that the S•••O chalcogen bond aids in achieving the requisite linear geometry for the SN2 reaction by aligning the AdoMet sulfur and methyl carbon atoms with the lysine ε-amine nucleophile. The loss of this interaction would potentially diminish the frequency with which this catalytically productive alignment occurs, modestly decreasing the reaction rate. To corroborate the experimental findings that S•••O chalcogen bonding enables SET7/9 to distinguish between AdoMet and AdoHcy, we performed QM calculations using active site models for the WT enzyme and N265A mutant (Figure 3 and Supplementary Figure 3). These models comprise residues forming an S•••O chalcogen bond and CH•••O hydrogen bonds to AdoMet. The AdoMet sulfonium cation and AdoHcy thioether groups were modeled as MeS+(Et)2 and S(Et)2 monomers, respectively.5 Two N-methyl acetamide (NMA) molecules were included in the models to mimic the backbone atoms of Val277 and His293, whose carbonyl groups form an NH•••O hydrogen bond to Asn265 and a CH•••O hydrogen bond to AdoMet, respectively. The side chain of Tyr335 that forms multiple CH•••O hydrogen bonds to AdoMet was represented as a 4-methylphenol molecule. The Cα atom and side chain of Asn265 were modeled as propanamide, whereas an ethyl group replaced the Ala265 side chain. A water molecule that forms a hydrogen bond with the Asn265 side chain (Figure 1A) was also included in the active site models. Constraints were applied to specific atoms in the models to ensure that the positions of these groups did not deviate from the crystallographic coordinates of WT SET7/9 and the N265A mutant during energy minimization. After generating the active site models for WT SET7/9 and the N265A mutant bound to MeS+(Et)2 and S(Et)2, pairwise interaction energies for the different monomers composing the complexes and the total interaction energy of each complex were calculated (Supplementary

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Table 2). In the course of analyzing the interactions among the various monomers, we noted that the propanamide is positioned to engage in an S•••O chalcogen bond and a methyl CH•••O hydrogen bond with the MeS+(Et)2 (Figure 1A and Figure 3). While it would be desirable to derive a precise energetic contribution of each of these two interactions separately, their dual presence and mutual interaction prevents a rigorous partition. Consistent with this observation, an energetic analysis of the propanamide and MeS+(Et)2 monomers illustrates that both the S•••O chalcogen bond and CH•••O hydrogen bond are essential contributors to the interaction between these groups, with the chalcogen bond displaying a greater apparent interaction energy (please see the Supplementary Text and Supplementary Figure 4). Utilizing the active site models of the WT enzyme and N265A mutant, we examined the energetic consequence of disrupting the S•••O chalcogen bond to AdoMet. The total binding energy of each complex was calculated by subtracting the sum of the individual energies of geometry-optimized monomers from the total interaction energy of each complex (Supplementary Table 2), as previously described.5 We then calculated the differences in the binding energies (ΔE) between WT SET7/9 and the N265A mutant for the MeS+(Et)2 and S(Et)2 complexes (Table 1). It is worth noting that the ΔE values reflect not only the interactions between the propanamide and the MeS+(Et)2 and S(Et)2 groups, but also the interactions of the propanamide with the surrounding active site residues. In addition, the N265A mutation modestly diminishes the thermodynamic stability of the mutant compared to the WT enzyme, thus precluding direct quantitative comparisons between the dissociation constants and the calculated binding energies. However, the trends in the binding energies for WT SET7/9 and the N265A mutant can be correlated with their respective binding affinities for AdoMet and AdoHcy. Consistent with prior calculations,13 there is the dramatic weakening in any sort of

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S•••O chalcogen or CH•••O hydrogen bond that occurs when the sulfonium cation is neutralized to a thioether, as evident in comparison of the pairwise interaction energies of propanamide and ethane with MeS+(Et)2 and S(Et)2 (Supplementary Table 2). In the case of the neutral S(Et)2, the pairwise interactions would attribute the stronger binding affinity of the WT enzyme versus the N265A mutant to the ability of the propanamide to engage in a hydrogen bond with 1) the NMA monomer mimicking the Val277 carbonyl group and 2) the active site water molecule (Figure 3 and Supplementary Figure 3). Together, these two hydrogen bonds account for 13 kcal mol-1 (Supplementary Table 2). However, the latter two interactions account for a similar amount in the case of the MeS+(Et)2, so one can attribute the differing binding behavior of WT SET7/9 and the N265A mutant with respect to AdoMet and AdoHcy almost completely to the combination S•••O chalcogen and CH•••O hydrogen bond between the AdoMet sulfonium cation and Asn265 carboxamide (Figure 3, Table 1, Supplementary Figure 3, and Supplementary Table 2). Based on these findings, we next examined whether Asn265 in SET7/9 is capable of recognizing AdoMet analogs that lack sulfonium or thioether groups. One such analog is sinefungin, a natural product inhibitor of AdoMet-dependent methyltransferases. In this inhibitor, the methyl and sulfonium groups of AdoMet are replaced by amine and methine (CH) moieties, respectively, thus eliminating S•••O chalcogen bonding. Surprisingly, the binding affinity of sinefungin is diminished by approximately 20-fold by the N265A substitution in SET7/9 (Table 1 and Supplementary Figure 5), indicating that this residue participates in inhibitor recognition. An examination of the crystal structure of SET7/9 bound to sinefungin22 illustrates that the Asn265 carboxamide is poised to form NH•••O and CH•••O hydrogen bonds with the amine and methine groups of the inhibitor, respectively (Figure 4A). These interactions mimic the CH•••O hydrogen bond and S•••O chalcogen bond formed with AdoMet in the

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enzyme active site (Figure 1A). A superimposition of the AdoMet- and sinefungin-bound complexes of SET7/9 reveals that the overall binding conformation of the substrate and inhibitor are structurally homologous, although there are subtle conformational differences between the amine and methine groups of sinefungin and the corresponding methyl and sulfonium moieties of AdoMet (Figure 4B). The amine group of the inhibitor is tilted toward the Asn265 carboxamide, permitting relatively short NH•••O and CH•••O hydrogen bonds with the carbonyl group. To gain further insight into the energetic basis of these interactions, we generated active site models for the WT enzyme and N265A mutant bound to sinefungin and calculated QM binding energies as described for AdoMet and AdoHcy (Supplementary Figure 6 and Supplementary Table 3). Sinefungin was modeled as 3-aminopentane to maintain isostericity with MeS+(Et)2. Binding energies were calculated with 3-aminopentane modeled in protonated ((Et)2CHNH3+) and deprotonated ((Et)2CHNH2) states, as its protonation state within the active site cannot be assumed a priori (Table 1). A comparison of the ΔE values reveals a substantial difference in binding energy for the (Et)2CHNH3+ monomer between WT SET7/9 and N265A mutant, whereas the difference for (Et)2CHNH2 is considerably smaller. This difference in the ΔE values is a consequence of the positive charge of the ammonium cation of (Et)2CHNH3+ that significantly augments the strengths of the NH•••O and the methine CH•••O hydrogen bonds to the propanamide group in comparison to the neutral (Et)2CHNH2 monomer, in agreement with prior calculations.23 These data corroborate the sinefungin dissociation constants for the WT SET7/9 and N265A mutant, demonstrating that Asn265 mediates high affinity binding of the inhibitor. In comparing AdoMet and sinefungin, the binding energy of the WT enzyme•MeS+(Et)2 complex (-56 kcal mol-1) falls between the binding energies of the SET7/9

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complexes with (Et)2CHNH3+ (-74 kcal mol-1) and (Et)2CHNH2 (-31 kcal mol-1). Given that SET7/9 binds AdoMet more tightly than sinefungin (Table 1), these binding energies suggest that sinefungin may bind within the active site of SET7/9 in an equilibrium that includes both protonated and deprotonated states of the amine group of the inhibitor. The findings that SET7/9 forms an S•••O chalcogen bond with AdoMet prompted us to investigate whether other AdoMet-dependent methyltransferases exhibit these interactions. An examination of the crystal structures of AdoMet-bound methyltransferases revealed several examples of these interactions in the canonical Rossmann-like fold, SPOUT, and SET domain KMT classes (Figure 5). S•••O chalcogen bonding to AdoMet frequently involves backbone peptide carbonyl groups, as illustrated in the structures of protein isoaspartyl methyltransferase, the SPOUT methyltransferase TrmD, and the KMT SUV420H2. In addition, side chains can participate in S•••O chalcogen bonding with AdoMet, as illustrated by Asn265 in SET7/9 and Tyr22 in the bacterial methyltransferase TylM1. Intramolecular chalcogen bonding can also occur when AdoMet adopts conformations that bring the O4 atom in its ribose ring into juxtaposition with the sulfur atom, as observed in dimethyladenosine transferase. Collectively, these structures illustrate that AdoMet S•••O chalcogen bonding is present in several methyltransferase classes and is not a unique feature of SET domain enzymes. Knowledge of S•••O chalcogen bonding in specific methyltransferases can be leveraged in structure-based design of AdoMet analog inhibitors that mimic these interactions within the active site. The substitution of the S•••O chalcogen bond and CH•••O hydrogen bond between Asn265 and AdoMet by the NH•••O and CH•••O hydrogen bonds with sinefungin exemplifies this strategy (Figure 4B).

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The propensity of certain methyltransferases to engage in S•••O chalcogen bonding with AdoMet shares interesting parallels with CH•••O hydrogen bonding to the substrate. The strengths of these interactions are considerably enhanced by electrostatic attraction to the AdoMet sulfonium cation compared to the neutral thioether of AdoHcy, consistent with QM studies.13, 23 Consequently, S•••O chalcogen and CH•••O hydrogen bonds may cooperatively contribute to AdoMet binding selectivity, allowing methyltransferases to discriminate between substrate and product. In addition, multivalent S•••O and CH•••O interactions with the AdoMet sulfonium cation (Figure 1A) appear to have catalytic roles in properly aligning the sulfur – methyl carbon bond with the nucleophile during SN2 methyl transfer reaction.4 Further studies are necessary to understand the contributions of these interactions in the reaction mechanism of AdoMet-dependent methyltransferases.

METHODS Protein Expression and Purification. The N265A mutation was introduced into the pHIS2 plasmid24 encoding SET7/9 (residues 110 – 366) using QuikChange mutagenesis and was sequence verified by dideoxy sequencing. The SET7/9 N265A mutant was purified as previously published, and co-purifying AdoMet was removed using a denaturation and refolding protocol.21 To increase the yield of AdoMet-free SET7/9 N265A for the ITC experiments with AdoMet and the TAF10 peptide, the protein (5 mg mL-1) was incubated with activated charcoal in a 1:2 weight ratio for 10 – 20 minutes at room temperature. The charcoal was removed by centrifugation and filtration, and the N265A mutant was further purified by gel filtration chromatography. Removal of the AdoMet from the protein was assessed using a Superdex

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Peptide column (GE Healthcare) under denaturing conditions by monitoring the absorbance at 255 nm. Protein concentration was measured at 280 nm in 6 M guanidinium chloride.

Crystallization and Structure Determination. Crystals of the SET7/9 N265A mutant bound to AdoHcy and the TAF10 K189me1 peptide were obtained by hanging drop vapor diffusion at 20 °C using a 1:1 ratio of the protein solution (4.0 – 8.0 mg/mL protein, 2.5 mM AdoMet, 2.0 mM TAF10 peptide, and 2.0 mM Tris(2-carboxyethyl)phosphine) and the crystallization solution (0.99 - 1.02 M sodium citrate, 12 - 22 mM NiCl2, and 100 mM imidazole pH 8.4), as previously described.4, 5, 25 Crystals were cryoprotected in 100 mM imidazole pH 8.2, 1.5 M sodium citrate, and 12 mM NiCl2 and flash-frozen in liquid nitrogen. Diffraction data were collected at the Advanced Photon Source Synchrotron beamline 21-ID-D (LS-CAT) and were processed and scaled using HKL2000.26 The structure was solved by molecular replacement using Phaser and the coordinates of a SET7/9 ternary complex (PDB accession code 4J83) as a search model.27 Model building was performed using Coot, and refinement and structure validation were performed using Phenix.28, 29 The final structure of the SET7/9 N265A mutant revealed a product ternary complex with AdoHcy and a TAF10-K189me1 peptide due to methylation of the TAF10 peptide by AdoMet. Structural figures were rendered using PyMOL (Schrödinger, LLC).

Biochemical Assays and Calorimetry. AdoMet was purified as previously published.21 Measurement of the TM value of the SET7/9 N265A mutant was performed as previously reported4 using 0.9 mg/mL protein in 20 mM sodium phosphate pH 7.0 and 100 mM NaCl with a temperature range of 10 – 100 °C and a scan rate of 0.5 °C/min at 3.0 atm constant pressure in a Nano DSC Calorimeter (TA Instruments). DSC data were processed using NanoAnalyze (TA

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Instruments) and Prism 6 (GraphPad). Binding experiments for the SET7/9 N265A mutant were performed in 20 mM sodium phosphate pH 7.0 and 100 mM NaCl. The dissociation constants for AdoMet, sinefungin, and the TAF10 peptide were determined by ITC at 20 °C using a MicroCal VP-ITC Calorimeter (Malvern Instruments). The dissociation constant of AdoMet was measured using 6.66 µM protein and 84.8 µM ligand, whereas the sinefungin dissociation constant was determined using 63.3 µM protein and 668 µM ligand. The TAF10 peptide dissociation constant was quantified as previously described.25 The dissociation constant of AdoHcy was measured using a previously reported tryptophan fluorescence binding assay with 1.75 µM SET7/9 N265A mutant and 0 to 9.26 mM AdoHcy in a Tecan Safire 2 microplate reader.4, 5 Steady state radiometric assays were performed in 100 mM Bicine pH 9.0 and 25 mM NaCl using 0.30 µM SET7/9 N265A mutant, 3H-methyl AdoMet (0.85 – 1440 µM) and 1.33 mM TAF10 biotinylated peptide Ac-SKSKDRKYTLT(K-EZLinkSSBiot)-amide (New England Peptide).4 Assays were run for two minutes at 37 °C in a final volume of 20 µl. The reactions were processed using Streptavidin resin (Genscript) to immobilize the biotinylated peptide, and the incorporation of the radiolabel into the peptide was quantified using liquid scintillation counting.

QM Calculations. All QM calculations were carried out with the density functional M06-2X in conjunction with the 6-31+G** basis set within the context of the Gaussian-09 software package. Binding energies of the complexes were evaluated as the difference between the energy of the complex and the sum of the energies of separately optimized monomers. In contrast to binding energies, interaction energies referenced the energies of monomer geometries within the entire complex. Both binding energies and interaction energies were corrected for basis sets

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superposition error using the counterpoise correction method. Optimizations were carried out with the restriction that certain atoms are held in their position within the experimental crystal structure.

Accession Codes The coordinates and structure factors for the SET7/9 N265AAdoHcyTAF10-K189me1 peptide complex (accession code 5EG2) have been deposited in the RCSB Protein Data Bank.

Acknowledgements We thank M. Luo for sharing the protocol for removing AdoMet bound to methyltransferases using activated charcoal and K. Wisser for her assistance with the DSC experiments. This work was supported by NSF-CHE-1508492 to R. Trievel. Use of the Advanced Photon Source, an Office of Science User Facility operated for the U.S. Department of Energy (DOE) Office of Science by Argonne National Laboratory, was supported by the U.S. DOE under Contract No. DE-AC02-06CH11357. Use of the LS-CAT Sector 21 was supported by the Michigan Economic Development Corporation and the Michigan Technology Tri-Corridor (Grant 085P1000817).

Supporting Information Available: This material is available free of charge via the Internet.

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KD (µM) WT AdoMet

0.053

AdoHcy

134

TAF10

4.9

Sinefungin

0.33

kcat (min-1)

29.6

KM (µM)

2.5 -1

-1

kcat/KM (µM min ) Complex energy +

12

N265A

± ± ± ±

0.003

± ± ±

0.9b

4.16

0.3

b

18.3

1.5

b

0.227

a

0.41

36b

301

0.1c

3.7

0.02a 7.46 Kinetic Parameters

± ± ± ±

0.03d

± ± ±

0.14

QM binding energy (kcal mol-1) WT N265A

18e 0.3d 0.07d

2.9 0.037 ΔE

MeS (Et)2 complex energy

-56.48

-35.43

-21.05

S(Et)2 complex energy

-19.61

-9.72

-9.89

3(Et)2CHNH3+ complex energy

-74.02

-44.85

-29.17

(Et)2CHNH2 complex energy

-30.96

-16.90

-14.06

Table 1: Binding and kinetic data, and QM energy calculations for WT SET7/9 and the N265A mutant. The KM and kcat/KM values are reported with respect to AdoMet. QM energies shown are the total binding energies of the active site models, minus the energy of the individual optimized monomer structures using M06-2X/6-31+G**. aThese data were originally published in (Horowitz et al., 2011)21; © the American Society for Biochemistry and Molecular Biology. b Reproduced from (Horowitz et al., 2013).4 cThese data were originally reported in (Del Rizzo et al., 2010)25; © the American Society for Biochemistry and Molecular Biology. dDissociation constants were measured by ITC. eDissociation constant was measured using an intrinsic tryptophan fluorescence assay.

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Figure 1: Crystal structure of the SET7/9 N265A mutant. (A) Active site of WT SET7/9 in complex with AdoMet and TAF10 K189A peptide (PDB accession code: 4J83). The S•••O chalcogen bond formed by the AdoMet sulfur atom and the Asn265 side chain is shown as purple dashes, and the hydrogen bond between the Asn265 amide and Val277 is shown in cyan. CH•••O hydrogen bonds to AdoMet are illustrated as orange dashes. (B) Structure of the SET7/9 N265A mutant bound to AdoHcy and a TAF10-K189me1 peptide. The alanine substitution is highlighted with slate blue carbon atoms. (C) Simulated annealing FO – FC omit map calculated for the active site of the N265A mutant. The map is contoured at 2.0 σ and is displayed for AdoHcy, TAF10 K189me1, Ala265, Val277, and the water molecule.

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Figure 2: Biochemical and kinetic characterization of the SET7/9 N265A mutant. (A) ITC analysis of AdoMet binding to the mutant. The top panel represents the titration of AdoMet into SET7/9 N265A. The bottom panel shows the curve fitted to the binding isotherm. (B) Tryptophan fluorescence assay of AdoHcy binding to SET7/9 N265A. Data points represent the averages from triplicate measurements, and the error bars represent two standard deviations. (C) Radiometric assay of SET7/9 N265A activity with a Michaelis-Menten curve fitted to the data. Data points represent the averages from triplicate measurements with the error bars representing standard error.

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Figure 3: Optimized geometry of a minimal active site model used in QM calculations to represent AdoMet, shown as MeS+(Et)2, bound to the active site of WT SET7/9. Distances are measured in Å, and asterisks are used to denote atoms that were frozen in the positions of the Xray coordinates.

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Figure 4: Comparison of sinefungin and AdoMet binding to WT SET7/9 (A) Active site of WT SET7/9 bound to sinefungin and an estrogen receptor α peptide (PDB accession code: 3CBP). Conventional hydrogen bonds are illustrated as cyan dashes, and CH•••O hydrogen bonds are shown as orange dashes. (B) Superimposition of the SET7/9 complexes with sinefungin and AdoMet from panel A and Figure 1A, respectively, aligned by their active site residues. Amino acids from the sinefungin and AdoMet complexes are denoted by light and dark gray carbon atoms, respectively. S•••O chalcogen bonding is depicted as magenta dashes.

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Figure 5: Examples of S•••O chalcogen bonding in AdoMet-dependent methyltransferases. Classes represented are the Rossmann-like fold (accession codes: 3PFG, 1JG4, and 1ZQ9), SPOUT (accession code: 1UAK), and SET domain (accession codes: 3RQ4 and 4J83) methyltransferases.30-33 The chalcogen bonds depicted exhibit S•••O interaction distances < 3.3 Å with a bond interaction angle > 150° (bond angle defined as O•••S-C, where S-C is the bond opposite the σ anti-bonding orbital).

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S•••O chalcogen bond between AdoMet and Asn265 in the lysine methyltransferase SET7/9. 39x34mm (300 x 300 DPI)


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