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Conservation and Functional Importance of Carbon-Oxygen Hydrogen Bonding in AdoMet-Dependent Methyltransferases Scott Horowitz, Lynnette M. A. Dirk, Joseph D Yesselman, Jennifer S Nimtz, Upendra Adhikari, Ryan A. Mehl, Steve Scheiner, Robert L. Houtz, Hashim M. Al-Hashimi, and Raymond C. Trievel J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/ja407140k • Publication Date (Web): 06 Sep 2013 Downloaded from http://pubs.acs.org on September 29, 2013
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Conservation and Functional Importance of Carbon-Oxygen Hydrogen Bonding in AdoMet-Dependent Methyltransferases Scott Horowitz1,2, Lynnette M.A. Dirk3, Joseph D. Yesselman2, Jennifer S. Nimtz4,5, Upendra Adhikari6, Ryan A. Mehl7, Steve Scheiner6, Robert L. Houtz3, Hashim M. Al-Hashimi2,4, Raymond C. Trievel5*
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Howard Hughes Medical Institute, Ann Arbor MI 48109 USA
Departments of Biophysics2, Chemistry4, and Biological Chemistry5, University of Michigan, Ann Arbor, MI 48109, USA 3
Department of Horticulture, University of Kentucky, Lexington, KY 40546 USA
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Department of Chemistry and Biochemistry, Utah State University, Logan, UT 84322 USA
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Department of Biochemistry and Biophysics, Oregon State University, Corvallis, OR 97331
USA
*Email:
[email protected] ACS Paragon Plus Environment
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Abstract: S-Adenosylmethionine (AdoMet)-based methylation is integral to metabolism and signaling. AdoMet-dependent methyltransferases belong to multiple distinct classes and share a catalytic mechanism that arose through convergent evolution; however, fundamental determinants underlying this shared methyl transfer mechanism remain undefined. A survey of high-resolution crystal structures reveals that unconventional carbon-oxygen (CH···O) hydrogen bonds coordinate the AdoMet methyl group in different methyltransferases irrespective of their class, active site structure, or cofactor binding conformation. Corroborating these observations, quantum chemistry calculations demonstrate that these charged interactions formed by the AdoMet sulfonium cation are stronger than typical CH···O hydrogen bonds. Biochemical and structural studies using a model lysine methyltransferase and an active site mutant that abolishes CH···O hydrogen bonding to AdoMet demonstrate that these interactions are important for high affinity AdoMet binding and transition state stabilization. Further, crystallographic and NMR dynamics experiments of the wild type enzyme demonstrate that the CH···O hydrogen bonds constrain the motion of the AdoMet methyl group, potentially facilitating its alignment during catalysis. Collectively, the experimental findings with the model methyltransferase and structural survey imply that methyl CH···O hydrogen bonding represents a convergent evolutionary feature of AdoMet-dependent methyltransferases, mediating a universal mechanism for methyl transfer.
INTRODUCTION S-Adenosylmethionine (AdoMet) is a versatile and ubiquitous enzyme substrate and is the most common methyl group donor in biology1,2. AdoMet-dependent methylation is essential to the metabolism of amino acids, cofactors, hormones, lipids, nucleic acids, and polysaccharides and has emerged as an important covalent modification of DNA and proteins, particularly with respect to epigenetic gene regulation. The enzymes that catalyze this reaction belong to as many as nine distinct classes that are unrelated by sequence, structure, or AdoMet binding modes3. Despite these differences, AdoMet-dependent methyltransferases share a conserved catalytic mechanism wherein the AdoMet methyl group is transferred to the acceptor substrate via an SN2 reaction1,4. This reaction requires a linear alignment of the acceptor substrate (nucleophile), methyl group (electrophile), and the sulfur atom (leaving group) of the product Sadenosylhomocysteine (AdoHcy)5. During the reaction, the methyl group inverts its stereochemistry, leading to a partially positively charged, sp2 planar transition state. Currently, 2
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there are primarily three models as to how methyltransferases achieve their catalytic power: active site compression6, selective transition state stabilization through hydrogen bonding and active site electrostatics7, and formation of a near attack conformation (NAC) between the electrophile and nucleophile5. It is conceivable that each of these mechanisms contributes at least in part to methyl transfer catalysis. Recently, unconventional CH···O hydrogen bonds were demonstrated to coordinate the AdoMet methyl group in the active site of the SET domain class of protein lysine methyltransferases using NMR spectroscopy8. These interactions are generally thought to contribute about half the strength of a conventional hydrogen bond9, although computational and experimental studies indicate that the energy of these interactions can vary greatly, and can have strengths equivalent to conventional hydrogen bonds, especially when the carbon is highly polarized or part of a charged group10,11. Although CH···O hydrogen bonding is ubiquitous in biomolecular structure and has been implicated in enzyme catalysis10-16, experimental evidence for the specific functions or importance of CH···O hydrogen bonding in biology is generally lacking. In the context of lysine methylation, these hydrogen bonds were hypothesized to perform several functions in lysine methyl transfer, including enhancing AdoMet binding, stabilizing the partially positively charged transition state, and aligning the methyl group for optimal transfer geometry14. Here, we present data using an array of biochemical and biophysical approaches demonstrating that CH···O hydrogen bonds greatly augment AdoMet binding affinity and transition state stability to a similar degree, and constrain the motion of the AdoMet methyl group, possibly contributing to NAC formation.
EXPERIMENTAL SECTION High-Resolution Crystal Structure PDB Survey All co-crystal structures of methyltransferases bound to AdoMet with resolution better than 2.00 Å were downloaded and visually inspected before inclusion in the AdoMet and protein methyl and methylene CH···O hydrogen bond surveys. Duplicate structures were removed, retaining the highest resolution structure of each methyltransferase. Electron density from the EDS server was examined to verify model quality when available17. Due to ambiguous 2Fo-Fc density corresponding to the AdoMet methyl group position, several structures (PDB accession codes 2IGT, 1FPX, 1I9G, 2EGV, 2QE6, 2YQZ, 3H6L, 3EEO, and 4DMG) were omitted from
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the survey. For certain structures (PDB accession codes 3UJ7, 1N2X, and 1ZQ9), analysis was restricted to a single copy of the methyltransferase, due to a lack of interpretable methyl group density. All other structures with multiple copies within the PDB file had CH···O hydrogen bonding acceptor parameters averaged before analysis. Accession codes 1N2X, 2ZUL and 1V2X were refined by real space parameters using Coot18 to adjust for model building and refinement anomalies before inclusion in the survey. In addition to AdoMet methyl and methylene groups, all protein side chain methyl and methylene C···O interactions were analyzed using identical hydrogen bonding cutoffs to provide a reference point for analyzing the properties of the AdoMet methyl and methylene CH···O hydrogen bonds. As methyl hydrogen positions for these structures were unknown, methyl group hydrogen bonding was based on van der Waals radii12 and non-hydrogen atom angles. Four separate criteria were used to determine if methyl groups participated in CH···O hydrogen bonding: C···O distance, X-C···O angle, and C···O-Y angle, where X and Y are the non-hydrogen atoms bonded to carbon and oxygen, respectively, and sp2 oxygen-methyl elevation angle19. Hydrogen bonding was determined using two scenarios 1) For C···O distances less than 3.25 Å, which is the sum of the van der Waals radii of carbon and oxygen; and 2) For distances greater or equal to 3.25 Å but less than 3.70 Å, which is the combined van der Waals radii of a C-H bond and oxygen arranged in linear geometry12. In the former scenario, methyl groups were considered to participate in CH···O hydrogen bonding if the C···O distance was less than 3.25 Å, the X-C···O angle was between 60° and 160°, and the elevation angle was less than 75°. For the latter scenario, methyl groups were considered to undergo CH···O hydrogen bonding if the C···O distance was less than 3.70 Å, the X-C···O angle was between 85° and 140°, and if elevation angle was less than 50°. The C···O-Y angle was required to be greater than 90° or between 75° and 150° for sp2 or sp3 hybridized oxygen atoms, respectively. In-house software to measure methyl hydrogen bonding criteria was written using MATCH API to manipulate atoms20. Based on this criterion, ~28% of the protein methyl groups surveyed exhibited CH···O hydrogen bonding. Protein methyl groups clearly favor relatively long CH···O interactions (mean of 3.8 Å), consistent with previous investigations of unpolarized methyl CH···O hydrogen bonding in small molecules21. Distance and angular distributions were volume-corrected by multiplying the counts by 1/r3 and 1/sinθ, respectively22. Distributions of angles and distances were analyzed using Origin (OriginLab).
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To calculate whether the AdoMet methyl CH···O hydrogen bonds were significantly different from that of protein methyl groups, we examined the average length of each of the two populations. The difference between the mean lengths of each population was determined to be 0.31 Å. To establish whether this difference is significant, we recombined both populations, and used the Fisher-Yates algorithm23 to randomly re-order the data set, followed by division into two subsets containing the same number of hydrogen bonding interactions as the two original populations (127 and 9881 members, respectively), and the difference between average lengths calculated. This process was repeated 100,000 times, generating a normal distribution with an average difference of 0.025 Å and standard deviation of 0.019 Å. These values yield a Z-score of 15.2σ, or a confidence value greater than 99.999%, permitting us to reject the null hypothesis. In addition to methyl groups, all AdoMet and protein methylene CH···O hydrogen bonds were measured and volume corrected as described above for methyl groups, with the exception that hydrogen atoms were modeled into the riding positions using Chimera24, and used to define CH···O hydrogen bond angles. In addition to the longer distance (
