Manipulating Unconventional CH-Based Hydrogen Bonding in a

Subscriber access provided by UQ Library

Letter

Manipulating Unconventional CH-based Hydrogen Bonding in a Methyltransferase via Non-Canonical Amino Acid Mutagenesis Scott Horowitz, Upendra Adhikari, Lynnette M. A. Dirk, Paul A Del Rizzo, Ryan A. Mehl, Robert L. Houtz, Hashim M. Al-Hashimi, Steve Scheiner, and Raymond C. Trievel ACS Chem. Biol., Just Accepted Manuscript • Publication Date (Web): 10 Jun 2014 Downloaded from http://pubs.acs.org on June 15, 2014

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

ACS Chemical Biology is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 18

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Chemical Biology

Manipulating Unconventional CH-based Hydrogen Bonding in a Methyltransferase via Non-Canonical Amino Acid Mutagenesis

Scott Horowitz1,2,3, Upendra Adhikari4, Lynnette M.A. Dirk5, Paul A. Del Rizzo6, Ryan A. Mehl7, Robert L. Houtz5, Hashim M. Al-Hashimi2,8,9, Steve Scheiner4, and Raymond C. Trievel6* 1

Howard Hughes Medical Institute, Ann Arbor MI 48109 USA

Departments of Biophysics2 and Molecular, Cellular, and Developmental Biology3, Biological Chemistry6, and Chemistry8, University of Michigan, Ann Arbor, MI 48109, USA 4

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

5

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

7

Department of Biochemistry and Biophysics, Oregon State University, Corvallis, OR 97331,

USA 9

Present address: Department of Biochemistry, Duke University, Durham, NC 27710, 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


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Abstract: Recent studies have demonstrated that the active sites of S-adenosylmethionine (AdoMet)-dependent methyltransferases form strong carbon-oxygen (CH···O) hydrogen bonds with the substrate’s sulfonium group that are important in AdoMet binding and catalysis. To probe these interactions, we substituted the non-canonical amino acid para-aminophenylalanine (pAF) for the active site tyrosine in the lysine methyltransferase SET7/9, which forms multiple CH···O hydrogen bonds to AdoMet and is invariant in SET domain enzymes. Using quantum chemistry calculations to predict the mutation’s effects, coupled with biochemical and structural studies, we observed that pAF forms a strong CH···N hydrogen bond to AdoMet that is offset by an energetically unfavorable amine group rotamer within the SET7/9 active site that hinders AdoMet binding and activity. Together, these results illustrate that the invariant tyrosine in SET domain methyltransferases functions as an essential hydrogen bonding hub and cannot be readily substituted by residues bearing other hydrogen bond acceptors.

2


Page 2 of 18

Page 3 of 18

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Text: AdoMet-dependent methylation is essential to numerous cellular processes involving metabolism and signal transduction. In this reaction, the methyl group is transferred from the AdoMet sulfonium cation to a nucleophilic acceptor atom via an SN2 reaction mechanism. Recent work suggests that unconventional CH···O hydrogen bonds1 are important in multiple aspects of this reaction, as AdoMet is capable of forming CH···O hydrogen bonds that are stronger than those typically observed in biological systems due to its cationic character2, 3. A crystallographic survey illustrated that the CH···O hydrogen bonds are conserved across different classes of AdoMet-dependent methyltransferases through apparent convergent evolution, suggesting that these interactions may be ideally suited to facilitating methyl transfer3. To investigate this possibility, we characterized the functions of CH···O hydrogen bonds between AdoMet and active site residue in the lysine methyltransferase SET7/9 and found that these interactions are important to substrate binding and electrostatic transition state stabilization, as well as restricting the motion of the AdoMet methyl group, presumably promoting catalysis3. Among these active site residues, the hydroxyl group of Tyr335, an invariant residue in the SET domain class, acts as an acceptor for many CH···O hydrogen bonds, in addition to donating a hydrogen bond to the backbone carbonyl group of Ala295 in SET7/9 (Figure 1A). Mutation of this residue to a phenylalanine (Y335F) severely impaired AdoMet binding affinity, demonstrating the importance of the CH···O hydrogen bonds to substrate recognition. Interestingly, the methyl transfer rate was not appreciably altered by this mutation, indicating that the Tyr335-mediated CH···O hydrogen bonding with AdoMet stabilizes the enzymesubstrate complex and transition states comparably. Building from this initial characterization, we sought to evaluate the relative importance of the OH···O and four CH···O hydrogen bonds

3



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

formed by Tyr335 in the SET7/9 active site (Figure 1A) to gain a more detailed understanding of the roles of CH···O hydrogen bonding in methylation. Toward this end, we undertook a combinatorial approach using quantum mechanical (QM) calculations, crystallography, and biochemistry with the goal of utilizing the QM calculations to model the active site energetics of wild type (WT) SET7/9 and Tyr335 mutants, which could then be directly compared with experimentally measured dissociation constants. This approach enabled us to rationally perturb the active site hydrogen bonding pattern and then analyze the resulting effects upon methyltransferase function. To introduce a different hydrogen bonding functionality into the active site, we employed an amber stop-codon suppression strategy to genetically substitute Tyr335 in SET7/9 with the non-canonical amino acid (ncAA) para-aminophenylalanine (pAF)4. This amino acid is isosteric to tyrosine but replaces the hydroxyl group with an amine moiety. We reasoned that pAF would act as a CH···N hydrogen bond acceptor to AdoMet, while simultaneously replacing the OH···O hydrogen bond of the WT enzyme with an NH···O hydrogen bond to the Ala295 carbonyl group, thus limiting alterations in the protein’s structure and stability. Although the Y335pAF mutant should be capable of accepting hydrogen bonds, it possesses one less lone pair of electrons compared to a tyrosine hydroxyl group, thus limiting the ability of pAF to act as a multi-furcated hydrogen bond acceptor. Conversely, the extra hydrogen atom in the pAF amine group would potentially introduce additional geometric and steric constraints within the SET7/9 active site. Prior to undertaking the QM calculations to predict the local effects of this mutation, we performed a series of control experiments to determine whether the SET7/9 Y335pAF mutant would be suitable for probing SET7/9 active site CH···O hydrogen bonding. Isothermal titration calorimetry experiments demonstrated the binding affinity of the Y335pAF mutant for a

4


Page 4 of 18

Page 5 of 18

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


substrate peptide representing the methylation site in the general transcription factor TAF10 was essentially identical to that of WT SET7/9 (Supplementary Figure 1A). In addition, differential scanning calorimetry illustrated that the WT enzyme and Y335pAF mutant exhibited essentially identical stabilities, consistent with the formation of a hydrogen bond between the Ala295 carbonyl group and pAF amine group, as well as the maintenance of the neutral charge on the Y335 side chain (Supplementary Figure 1B and Supplementary Text). To further examine the effects of this mutation on the enzyme’s overall and active site structures, we determined its cocrystal structure in complex with AdoHcy and a TAF10 peptide at 1.6 Å resolution (Figure 1 and Supplementary Table 1)5. The coordinates of this complex and the analogous WT complex superimpose with a Cα RMSD of 0.14 Å, illustrating that the WT enzyme and mutant are structurally homologous. Further, the structural hydrogen bond between Tyr335 and the carbonyl group of Ala295 is preserved by the pAF substitution (Figure 1B). The conventional hydrogen bonds between the enzyme and its ligands, AdoHcy and the TAF10 peptide, also appear to be preserved, as are the overall conformations the active site residues compared with the WT enzyme. Together, these results demonstrate that the SET7/9 Y335pAF mutant retains the structure and protein substrate binding properties of the WT enzyme. To develop a molecular-level understanding of the effect of changing the Tyr335 hydrogen bonding pattern, QM calculations were performed to model the effects of substitutions at the Tyr335 position on ligand binding. We began with a comparison of the energetics of binding a sulfonium cation mimic of AdoMet, MeS+(Et)2, to that of a thioether mimic of AdoHcy, S(Et)2. Notably, the sulfur atom of AdoHcy is a neutral thioether moiety due to the loss of the methyl group, unlike the sulfonium cation of AdoMet. Due to the highly homologous AdoMet and AdoHcy binding modes in the WT enzyme and Tyr335 mutants, which is reflected

5



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

in the calculations (see below), trends in the difference in binding affinities of AdoHcy and AdoMet between the different mutants should be well represented by calculations that focus on the active site. Completing the model active site using the crystal structure of SET7/9, a phenol molecule was inserted in the position of Tyr335, and an N-methylacetamide (NMA) molecule was placed in the position of Ala295 so as to simulate the hydrogen bonding of its peptide group. The adenine group of the cofactors was also included to account for the CH···O hydrogen bond between its C8 atom and the Tyr335 hydroxyl group (Supplementary Figure 2A-B). Since a full geometry optimization of this four-unit model, without the restraints of the rest of the system, would likely take the geometry far afield from the structure in the enzyme, several selected atoms were fixed in their X-ray positions during geometry optimizations (Supplementary Figure 3; please see the supplementary text for a discussion of model parameters and dielectric). The total binding energy was calculated as the difference in energy between the optimized complex and the sum of the energies of the four subunits, each with its geometry optimized separately. This quantity reflects not only the direct interaction of the AdoMet, or other moiety, with the active site, but also any changes induced by the AdoMet on the three other groups in the site. This total binding energy can also be decomposed into pairwise terms that reflect the strength of the interaction between any given pair of subunits to assist in the analysis. This quantity was computed first for the AdoHcy and AdoMet systems, and then for the Y335pAF and Y335F mutants in which the phenol group in the WT complex was substituted with an aniline and a benzene molecule, respectively (Figure 2 and Supplementary Figure 2C-D). Examining the wild type substrate and product models, the active site energy is more favorable for the MeS+(Et)2-containing complex compared to S(Et)2 complex by 11 kcal mol-1, consistent with past experiments (Table 1)3, 6. Considering the pairwise energies, this preference

6


Page 6 of 18

Page 7 of 18

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


arises due to the strong attractive interaction of the NMA and Tyr335 groups with the positively charged AdoMet (Supplementary Table 2). In contrast, in the benzene and aniline mutations, the selectivity for as MeS+(Et)2 over S(Et)2 is dramatically reduced (Table 1). This loss of selectivity is similar in magnitude between the two mutants. However, analyzing the pairwise interactions in the two mutants, the origin of the loss of binding energy differential is clearly different in each case (Supplementary Table 2). In the case of the phenylalanine mutant, the primary cause for the loss in selectivity is the abrogation of the Tyr335-mediated hydrogen bonds, similar to our previous interpretation3. In contrast, the aniline group forms a strong CH···N hydrogen bond to the sulfonium group. The loss in binding energy in the aniline case instead stems primarily from a change in conformation of the aniline amine group. To accommodate the methyl group and simultaneously form the aforementioned CH···N hydrogen bond, the amine group rotates 58° about the plane of aniline ring (compared to the optimized monomer) into a less energetically favorable position (Figure 2). Accordingly, there is an energetic penalty incurred by optimizing hydrogen bonding in the active site, thus sacrificing the lowest-energy aniline amine conformation in order to better accommodate CH···N hydrogen bonding to AdoMet. Additionally, the limited angular range of the amine group compared with the hydroxyl moiety to act as a hydrogen bond acceptor eliminates the ability for the aniline amine group to simultaneously serve as an effective CH···N hydrogen bond acceptor to the C8 atom of the adenine group (Supplementary Table 2), further reducing the selectivity between the two ligandbound states. Consequently, by optimizing the geometry of the CH···N hydrogen bond to AdoMet, the NH···O hydrogen bond to the Ala295 carbonyl group is correspondingly weakened, leading to a lower overall binding energy (Supplementary Table 2).

7



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

To test the QM predictions, we examined whether the Y335pAF mutation impaired cofactor binding or catalytic efficiency. The AdoMet and AdoHcy binding affinities of the SET7/9 Y335pAF mutant were comparable in magnitude, but were diminished by 10,000-fold and six-fold, respectively, compared to the WT enzyme, suggesting that the loss of the tyrosine hydroxyl group abolished selectivity for the substrate over the product, consistent with the QM calculations (Table 1 and Figure 3A-B). Despite the dramatic loss in binding affinity, the change in catalytic rate of the mutant was comparatively small. The kcat value Y335pAF was reduced ~35-fold relative to WT (Figure 3C). In terms of transition state stabilization, this change amounts to a 2.2 kcal mol-1, or 10% increase, of the activation barrier for methyl transfer. Of note, methyl transfer has been shown to be at least partially rate-limiting in SET7/93, 7. Accordingly, substituting CH···O hydrogen bonding of the Tyr335 hydroxyl group in the WT enzyme to a CH···N hydrogen bond in the Y3335pAF mutant did not substantially alter the activation barrier for catalysis. Through substituting the hydroxyl group of Tyr335 with an amine group using the ncAA pAF, we replaced the CH···O hydrogen bond accepting capacity of this residue with a CH···N hydrogen bond. However, the increased restrictions on the geometry of this position were detrimental to the active site when bound to AdoMet, as the cost of the rearrangement of the aniline amine group substantially decreased AdoMet binding affinity. As a correlating rearrangement was unnecessary to bind the product AdoHcy, the selectivity of the SET7/9 active site toward AdoMet versus AdoHcy was substantially decreased. Continuing this trend, no defect in melting temperature was observed for the mutant, suggesting that in the free state, the pAF amine group is free to optimize its geometry and hydrogen bonding to the carbonyl group of Ala295 without restriction (Supplementary Figure 1B). These results illustrate that the Tyr335

8


Page 8 of 18

Page 9 of 18

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


hydroxyl group functions as a hydrogen bonding hub in the SET7/9 active site. Its ability to act as a multi-furcated hydrogen bond acceptor, without imposing its own steric or geometrical constraints on the active site, is essential to preferential recognition for AdoMet over AdoHcy. This observation is consistent with the structural homology of the invariant tyrosine in the active sites of SET domain methyltransferases, and its conserved CH···O hydrogen bonding to AdoMet (Supplementary Figure 4)3. Given this conservation, the invariant tyrosine conceivably functions as a hydrogen bonding hub throughout the SET domain class. Additionally, inspection other methyltransferase class structures reveals active site tyrosines that engage in CH···O hydrogen bonding with AdoMet3. For example, in the Rossmann fold methyltransferase TylMl8, Tyr14 forms a CH···O hydrogen bond with the AdoMet methyl group (Supplementary Figure 4). The active site of TylM1 differs substantially from that of SET7/9, implying altered CH···O hydrogen bonding strength and therefore potential functional differences. Future studies are needed to ascertain how the properties of tyrosine-mediated CH···O hydrogen bonds vary across different methyltransferase classes. The QM calculations yielded another unanticipated observation about the SET7/9 active site; specifically, the electrostatic repulsion between the AdoMet sulfonium cation and the C8 position of adenine, the most acidic carbon atom in the purine ring system. Removing the charge from the sulfonium cation alleviated this repulsion. Notably, this conformation is dissimilar to that found in solution9 and in other methyltransferase classes10. This observation raises the intriguing possibility that the different AdoMet binding conformations in various methyltransferase classes may serve to tune the substrate’s methyl transfer reactivity and merits further investigation.

9



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 10 of 18

METHODS Protein Expression and Purification. The gene encoding the catalytic domain of SET7/9 (110– 366) was subcloned into the pBAD vector, followed by site-directed mutagenesis to convert Tyr335 to an amber stop codon for introduction of the ncAA, pAF. SET7/9 Y335pAF was recombinantly expressed in DH10B E. coli containing pDule-pAF vector according to previously published protocol for genetic incorporation of ncAAs11. Addition of 1.0 mM L-pAF during induction yielded 100% labeled protein at half the yield of the WT enzyme as judged by SDSPAGE. The remaining enzyme was truncated at residue 334. Induction without pAF yielded only SET7/9 truncated at residue 334. SET7/9 Y335pAF was purified as previously described12, with the exception that gel filtration chromatography was performed using a HiLoad Superdex 75 column (GE Healthcare) for the pAF mutant. This chromatography effectively separated the SET7/9 Y335pAF from the truncated enzyme based on the difference in their elution profiles, permitting the latter to be eliminated as a contaminant.

X-ray Crystallography and Biochemical Assays. Y335F SET7/9 crystals were grown by hanging drop vapor diffusion as previously described3, 5 (Supplementary Table 1), in which the crystallization solution was mixed in a 1:1 ratio with 10-12 mg mL-1 SET7/9, 3.0–4.5 mM AdoHcy, and 2.0 mM of a 10-residue peptide of TAF10 peptide in 20 mM TRIS pH 8.0, 100 mM NaCl and 2.0 mM Tris(2-carboxyethyl)phosphine. Crystallization solutions contained 0.861.07 M sodium citrate with 100 mM imidazole, pH 7.6–8.4 and 8.0–18 mM NiCl2. Crystals were flash frozen in 1.5 M sodium citrate, with 1.0 mM AdoHcy, 10–13 mM NiCl2 and 100 mM imidazole pH 8.2–8.7. Diffraction data were collected at the Advanced Photon Source Synchrotron beamline 21-IDG (LS-CAT), at 100 K and wavelength 0.9786 nm, and were

10


Page 11 of 18

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


processed using HKL200013. The structures were solved by molecular replacement using MOLREP14, with the previously reported protein coordinates of the SET7/9 ternary complex (PDB accession code 3M53)5 as the search model. Model building and refinement were carried out using Coot15, MiFit (version 2010.10 http://code.google.com/p/mifit), and REFMAC16. The quality of the final model was verified by Molprobity17. CNS was used to generate simulated annealing omit maps18, with K189 of the TAF10 peptide, pAF and AdoHcy omitted. RMSD values were calculated and structural figure rendered in PyMol (Schrödinger, LLC). Radiometric methyltransferase kinetics assays (using 10 µM Y335pAF mutant), tryptophan fluorescence binding experiments, ITC, DSC, were performed as described previously3.

QM Calculations. All the quantum mechanical calculations were carried out via Gaussian-09 package19. Density functional theory with M06-2X variant of functional20 was used with the 631+G** basis set. This level of theory has been found to be in good agreement with experimental values and MP2/aug-cc-pVDZ level of theory2, 21, 22. Binding energies were calculated as the difference between the energy of the complex and sum of the optimized monomers, and were corrected for basis set superposition error using the counterpoise procedure. Structural figures were rendered in Chemcraft (Chemcraft).

Supporting Information Additional discussion of the protonation status of residue 335 in SET7/9, QM modeling of the SET7/9 active site, effect of the dielectric constant on selectivity, and supplementary data tables and figures. This material is available free of charge via the Internet at http://pubs.acs.org.

11



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 12 of 18

Accession Codes The coordinates and structure factors for the SET7/9 Y335pAFAdoHcyTAF10 peptide complex (accession code 4J7F) have been deposited in the RCSB Protein Data Bank.

KD (µM) Y335pAF

WT a

AdoMet

0.053 b

AdoHcy

134

c

TAF10

4.9

± 0.003

980

± 36

52

± 26

714

± 197

b

± 0.3

± 210

840

± 0.1

Y335F b b

± 170

3.1

± 0.2

8.8

Kinetic Parameters WT b

-1

kcat (min )

29.6 b

KM (µM) -1

kcat/KM (µM min )

b

Y335pAF ± 0.9

0.84

2.5

± 0.3

330

12.0

± 1.5

0.0026

± 0.06

Y335F b

± 60 ± 0.0005

45.2

b b

± 3.6

150

± 30

0.31

± 0.068

-1

QM Energy Calculations (kcal mol ) Phenol

Aniline

Benzene

MeS (Et)2 Complex Energy

-27.9

-16.1

-10.8

S(Et)2 Complex Energy

-16.9

-10.1

-5.5

+

Table 1: Binding and kinetic data, and QM energy calculations of WT SET7/9 and Tyr335 mutants. The KM and kcat/KM values are reported with respect to AdoMet. QM energies shown are the total binding energies of the model complexes, minus the energy of the individual optimized monomer structures using M06-2X/6-31+G**. a,cThis research was originally published in a23 and c5; © the American Society for Biochemistry and Molecular Biology. cReproduced from3.

12


Page 13 of 18

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 1: Structure of WT SET7/9 and Y335pAF mutant bound to AdoHcy and TAF10 peptide. (A) WT SET7/9 bound to AdoMet and TAF10 K189A (4J83.pdb). CH···O hydrogen bonds formed by AdoMet and the OH···O hydrogen bond between Ala295 are denoted by orange, and cyan dashes, respectively3. All four CH···O hydrogen bonds accepted by the hydroxyl group of Tyr335 are probed by the pAF mutation (B) Active site of the SET7/9 Y335pAF mutant (4J7F.pdb). The NH···O hydrogen bond between Ala295 and pAF335 and van der Waals contact between pAF335 and the AdoHcy adenine group are denoted by cyan and magenta dashes, respectively. (C) Simulated annealing omit map the of SET7/9 Y335pAF active site contoured at 2.0 σ.

13



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 2: QM calculations of aniline ring in the SET7/9 Y335pAF active site. Active sites of (A and C) MeS+(Et)2 and (B and D) S(Et)2, depicting the rotation of the amine group by 58° compared with the (E) optimized monomer aniline structure.

14


Page 14 of 18

Page 15 of 18

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 3: Binding and methyltransferase assays of SET7/9 Y335pAF. Fluorescence binding assays of (A) AdoHcy and (B) AdoMet with SET7/9 Y335pAF. (C) Radiometric methyltransferase assay and Michaelis-Menten fit for the SET7/9 Y335pAF mutant.

Acknowledgements This work was supported by NSF-MCB-0448297 to R. Mehl, by grants from the University of Michigan’s Biomedical Research Council and the Office for the Vice President for Research and NSF (CHE-1213484) to R. Trievel, the Kentucky Agriculture Experiment station Hatch Project #KY011031 to R. Houtz and L. Dirk, and NSF-CHE-1026826 to S. Scheiner. 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 TriCorridor (Grant 085P1000817).

15



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 16 of 18

References [1] Horowitz, S., and Trievel, R. C. (2012) Carbon-oxygen hydrogen bonding in biological structure and function, J Biol Chem 287, 41576-41582. [2] Adhikari, U., and Scheiner, S. (2013) Magnitude and mechanism of charge enhancement of CH..O hydrogen bonds, J Phys Chem A 117, 10551-10562. [3] Horowitz, S., Dirk, L. M., Yesselman, J. D., Nimtz, J. S., Adhikari, U., Mehl, R. A., Scheiner, S., Houtz, R. L., Al-Hashimi, H. M., and Trievel, R. C. (2013) Conservation and functional importance of carbon-oxygen hydrogen bonding in AdoMet-dependent methyltransferases, J Am Chem Soc 135, 15536-15548. [4] Mehl, R. A., Anderson, J. C., Santoro, S. W., Wang, L., Martin, A. B., King, D. S., Horn, D. M., and Schultz, P. G. (2003) Generation of a bacterium with a 21 amino acid genetic code, J Am Chem Soc 125, 935-939. [5] Del Rizzo, P. A., Couture, J. F., Dirk, L. M. A., Strunk, B. S., Roiko, M. S., Brunzelle, J. S., Houtz, R. L., and Trievel, R. C. (2010) SET7/9 Catalytic Mutants Reveal the Role of Active Site Water Molecules in Lysine Multiple Methylation, J Biol Chem 285, 3184931858. [6] Couture, J. F., Hauk, G., Thompson, M. J., Blackburn, G. M., and Trievel, R. C. (2006) Catalytic roles for carbon-oxygen hydrogen bonding in SET domain lysine methyltransferases, J Biol Chem 281, 19280-19287. [7] Ibanez, G., McBean, J. L., Astudillo, Y. M., and Luo, M. K. (2010) An enzyme-coupled ultrasensitive luminescence assay for protein methyltransferases, Anal Biochem 401, 203210. [8] Carney, A. E., and Holden, H. M. (2011) Molecular architecture of TylM1 from Streptomyces fradiae: an N,N-dimethyltransferase involved in the production of dTDPD-mycaminose, Biochemistry 50, 780-787. [9] Markham, G. D., and Reczkowski, R. S. (2004) Structural studies of inhibition of Sadenosylmethionine synthetase by slow, tight-binding intermediate and product analogues, Biochemistry 43, 3415-3425. [10] Schubert, H. L., Blumenthal, R. M., and Cheng, X. D. (2003) Many paths to methyltransfer: a chronicle of convergence, Trends Biochem Sci 28, 329-335. [11] Hammill, J. T., Miyake-Stoner, S., Hazen, J. L., Jackson, J. C., and Mehl, R. A. (2007) Preparation of site-specifically labeled fluorinated proteins for 19F-NMR structural characterization, Nat Protoc 2, 2601-2607. [12] Trievel, R. C., Beach, B. M., Dirk, L. M. A., Houtz, R. L., and Hurley, J. H. (2002) Structure and catalytic mechanism of a SET domain protein methyltransferase, Cell 111, 91-103. [13] Otwinowski, Z., and Minor, W. (1997) Processing of X-ray diffraction data collected in oscillation mode, Method Enzymol 276, 307-326. [14] Vagin, A., and Teplyakov, A. (2000) An approach to multi-copy search in molecular replacement, Acta Crystallogr D 56, 1622-1624. [15] Emsley, P., Lohkamp, B., Scott, W. G., and Cowtan, K. (2010) Features and development of Coot, Acta Crystallogr D 66, 486-501. [16] Vagin, A. A., Steiner, R. A., Lebedev, A. A., Potterton, L., McNicholas, S., Long, F., and Murshudov, G. N. (2004) REFMAC5 dictionary: organization of prior chemical knowledge and guidelines for its use, Acta Crystallogr D 60, 2184-2195.

16


Page 17 of 18

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


[17] Chen, V. B., Arendall, W. B., Headd, J. J., Keedy, D. A., Immormino, R. M., Kapral, G. J., Murray, L. W., Richardson, J. S., and Richardson, D. C. (2010) MolProbity: all-atom structure validation for macromolecular crystallography, Acta Crystallogr D 66, 12-21. [18] Brunger, A. T., Adams, P. D., Clore, G. M., DeLano, W. L., Gros, P., Grosse-Kunstleve, R. W., Jiang, J. S., Kuszewski, J., Nilges, M., Pannu, N. S., Read, R. J., Rice, L. M., Simonson, T., and Warren, G. L. (1998) Crystallography & NMR system: A new software suite for macromolecular structure determination, Acta Crystallogr D 54, 905921. [19] Frisch, M. J., Trucks, G. W., Schlegel, H. B., Scuseria, G. E., Robb, M. A., Cheeseman, J. R., Scalmani, G., Barone, V., Mennucci, B., Petersson, G. A., Nakatsuji, H., Caricato, M., Li, X., Hratchian, H. P., Izmaylov, A. F., Bloino, J., Zheng, G., Sonnenberg, J. L., Hada, M., Ehara, M., Toyota, K., Fukuda, R., Hasegawa, J., Ishida, M., Nakajima, T., Honda, Y., Kitao, O., Nakai, H., Vreven, T., Montgomery, J., J. A., Peralta, J. E., Ogliaro, F., Bearpark, M., Heyd, J. J., Brothers, E., Kudin, K. N., Staroverov, V. N., Kobayashi, R., Normand, J., Raghavachari, K., Rendell, A., Burant, J. C., Iyengar, S. S., Tomasi, J., Cossi, M., Rega, N., Millam, J. M., Klene, M., Knox, J. E., Cross, J. B., Bakken, V., Adamo, C., Jaramillo, J., Gomperts, R., Stratmann, R. E., Yazyev, O., Austin, A. J., Cammi, R., Pomelli, C., Ochterski, J. W., Martin, R. L., Morokuma, K., Zakrzewski, V. G., Voth, G. A., Salvador, P., Dannenberg, J. J., Dapprich, S., Daniels, A. D., Farkas, O., Foresman, J. B., Ortiz, J. V., Cioslowski, J., and Fox, D. J. (2009) Gaussian 09, Revision B.01 ed., Wallingford, CT. [20] Zhao, Y., and Truhlar, D. G. (2006) Comparative DFT study of van der waals complexes: Rare-gas dimers, alkaline-earth dimers, zinc dimer, and zinc-rare-gas dimers, J Phys Chem A 110, 5121-5129. [21] Vincent, M. A., and Hillier, I. H. (2011) The structure and interaction energies of the weak complexes of CHClF2 and CHF3 with HCCH: a test of density functional theory methods, Phys Chem Chem Phys 13, 4388-4392. [22] Majumder, M., Mishra, B. K., and Sathyamurthy, N. (2013) CH center dot center dot center dot pi and pi center dot center dot center dot pi interaction in benzene-acetylene clusters, Chem Phys Lett 557, 59-65. [23] Horowitz, S., Yesselman, J. D., Al-Hashimi, H. M., and Trievel, R. C. (2011) Direct evidence for methyl group coordination by carbon-oxygen hydrogen bonds in the lysine methyltransferase SET7/9, J Biol Chem 286, 18658-18663.

17



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

101x101mm (300 x 300 DPI)


Page 18 of 18

Manipulating Unconventional CH-Based Hydrogen Bonding in a

Recommend Documents