Direct Evidence for Methyl Group Coordination by Carbon-Oxygen Hydrogen Bonds in the Lysine Methyltransferase SET7/9
2011; Elsevier BV; Volume: 286; Issue: 21 Linguagem: Inglês
10.1074/jbc.m111.232876
ISSN1083-351X
AutoresScott Horowitz, Joseph D. Yesselman, Hashim M. Al‐Hashimi, Raymond C. Trievel,
Tópico(s)RNA modifications and cancer
ResumoSET domain lysine methyltransferases (KMTs) are S-adenosylmethionine (AdoMet)-dependent enzymes that catalyze the site-specific methylation of lysyl residues in histone and non-histone proteins. Based on crystallographic and cofactor binding studies, carbon-oxygen (CH···O) hydrogen bonds have been proposed to coordinate the methyl groups of AdoMet and methyllysine within the SET domain active site. However, the presence of these hydrogen bonds has only been inferred due to the uncertainty of hydrogen atom positions in x-ray crystal structures. To experimentally resolve the positions of the methyl hydrogen atoms, we used NMR 1H chemical shift coupled with quantum mechanics calculations to examine the interactions of the AdoMet methyl group in the active site of the human KMT SET7/9. Our results indicated that at least two of the three hydrogens in the AdoMet methyl group engage in CH···O hydrogen bonding. These findings represent direct, quantitative evidence of CH···O hydrogen bond formation in the SET domain active site and suggest a role for these interactions in catalysis. Furthermore, thermodynamic analysis of AdoMet binding indicated that these interactions are important for cofactor binding across SET domain enzymes. SET domain lysine methyltransferases (KMTs) are S-adenosylmethionine (AdoMet)-dependent enzymes that catalyze the site-specific methylation of lysyl residues in histone and non-histone proteins. Based on crystallographic and cofactor binding studies, carbon-oxygen (CH···O) hydrogen bonds have been proposed to coordinate the methyl groups of AdoMet and methyllysine within the SET domain active site. However, the presence of these hydrogen bonds has only been inferred due to the uncertainty of hydrogen atom positions in x-ray crystal structures. To experimentally resolve the positions of the methyl hydrogen atoms, we used NMR 1H chemical shift coupled with quantum mechanics calculations to examine the interactions of the AdoMet methyl group in the active site of the human KMT SET7/9. Our results indicated that at least two of the three hydrogens in the AdoMet methyl group engage in CH···O hydrogen bonding. These findings represent direct, quantitative evidence of CH···O hydrogen bond formation in the SET domain active site and suggest a role for these interactions in catalysis. Furthermore, thermodynamic analysis of AdoMet binding indicated that these interactions are important for cofactor binding across SET domain enzymes. IntroductionPost-translational modifications in proteins are now well recognized as important players in many biological processes. Among these modifications, site-specific lysine methylation by SET domain KMTs 3The abbreviations used are: KMT, lysine methyltransferase; AdoMet, S-adenosylmethionine; CH···O, carbon-oxygen hydrogen bond; B3LYP, Becke, three-parameter, Lee-Yang-Parr; MD, molecular dynamics; HSQC, heteronuclear single quantum coherence; ITC, isothermal titration calorimetry; AdoHcy, S-adenosylhomocysteine; LSMT, Rubisco large subunit methyltransferase. is known to be critical to a diverse set of processes within the nucleus, including gene expression, cell cycle progression, and DNA damage response (1Albert M. Helin K. Semin. Cell Dev. Biol. 2010; 21: 209-220Crossref PubMed Scopus (228) Google Scholar, 2Dillon S.C. Zhang X. Trievel R.C. Cheng X.D. Genome Biol. 2005; 6: 227Crossref PubMed Scopus (547) Google Scholar). In particular, the human KMT SET7/9 has been shown to methylate lysine residues on many histone and non-histone proteins and is now considered to be important in many cellular pathways (3Pradhan S. Chin H.G. Esteve P.O. Jacobsen S.E. Epigenetics. 2009; 4: 282-285Crossref Scopus (103) Google Scholar). Furthermore, SET7/9 has emerged as an archetype for the specificity and catalytic mechanism of the SET domain family due to multiple high resolution crystal structures, NMR analyses, and computational studies on its structure and function (4Kwon T. Chang J.H. Kwak E. Lee C.W. Joachimiak A. Kim Y.C. Lee J. Cho Y. EMBO J. 2003; 22: 292-303Crossref PubMed Scopus (105) Google Scholar, 5Xiao B. Jing C. Wilson J.R. Walker P.A. Vasisht N. Kelly G. Howell S. Taylor I.A. Blackburn G.M. Gamblin S.J. Nature. 2003; 421: 652-656Crossref PubMed Scopus (302) Google Scholar, 6Couture J.F. Hauk G. Thompson M.J. Blackburn G.M. Trievel R.C. J. Biol. 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U.S.A. 2010; 107: 6817-6822Crossref PubMed Scopus (31) Google Scholar) are difficult to employ for many proteins, including SET7/9. However, NMR spectroscopy holds promise in identifying CH···O hydrogen bonds in proteins and other macromolecules via 1H chemical shift (23Ash E.L. Sudmeier J.L. Day R.M. Vincent M. Torchilin E.V. Haddad K.C. Bradshaw E.M. Sanford D.G. Bachovchin W.W. Proc. Natl. Acad. Sci. U.S.A. 2000; 97: 10371-10376Crossref PubMed Scopus (128) Google Scholar, 36Scheiner S. J. Phys. Chem. B. 2009; 113: 10421-10427Crossref PubMed Scopus (36) Google Scholar, 37Yates J.R. Pham T.N. Pickard C.J. Mauri F. Amado A.M. Gil A.M. Brown S.P. J. Am. Chem. Soc. 2005; 127: 10216-10220Crossref PubMed Scopus (171) Google Scholar).Recent structural and functional studies have suggested that CH···O hydrogen bonds play pivotal roles in substrate binding and catalysis in SET domain KMTs (6Couture J.F. Hauk G. Thompson M.J. Blackburn G.M. Trievel R.C. J. Biol. Chem. 2006; 281: 19280-19287Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 38Couture J.F. Dirk L.M. Brunzelle J.S. Houtz R.L. Trievel R.C. Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 20659-20664Crossref PubMed Scopus (89) Google Scholar, 39Del Rizzo P.A. Couture J.F. Dirk L.M. Strunk B.S. Roiko M.S. Brunzelle J.S. Houtz R.L. Trievel R.C. J. Biol. Chem. 2010; 285: 31849-31858Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). These interactions were identified in x-ray crystal structures, in which C···O distances between the AdoMet methyl carbon, structurally conserved carbonyl oxygen atoms, and an invariant tyrosine were appropriate (<3.7 Å) for CH···O hydrogen bonding to occur (6Couture J.F. Hauk G. Thompson M.J. Blackburn G.M. Trievel R.C. J. Biol. Chem. 2006; 281: 19280-19287Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar) (supplemental Fig. S1). These hydrogen bonds can form because the electron withdrawing character of the sulfonium cation polarizes the methyl group of AdoMet. Isothermal calorimetry demonstrated that AdoMet displays higher binding affinity to a SET domain KMT than AdoMet analogues that are incapable of forming CH···O hydrogen bonds (6Couture J.F. Hauk G. Thompson M.J. Blackburn G.M. Trievel R.C. J. Biol. Chem. 2006; 281: 19280-19287Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar), indicating the importance of these interactions in substrate binding. In addition, it was postulated that the CH···O hydrogen bonds align the AdoMet methyl group in the requisite linear geometry with the substrate lysine ϵ-amine group for the SN2 methyl transfer reaction. (supplemental Fig. S2) (2Dillon S.C. Zhang X. Trievel R.C. Cheng X.D. Genome Biol. 2005; 6: 227Crossref PubMed Scopus (547) Google Scholar, 5Xiao B. Jing C. Wilson J.R. Walker P.A. Vasisht N. Kelly G. Howell S. Taylor I.A. Blackburn G.M. 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Here, we address the fundamental question, is there direct experimental evidence for CH···O hydrogen bonding between the AdoMet methyl group and the SET domain active site in solution?RESULTSTo probe CH···O hydrogen bonding between SET7/9 and the AdoMet methyl group, we examined the NMR chemical shift produced by the AdoMet methyl group while bound to SET7/9. To measure the bound 1H chemical shift for the AdoMet methyl group and distinguish it from resonances arising from the enzyme, we synthesized [13C]methyl-labeled AdoMet using AdoMet synthase and [methyl-13C]methioine (43Iwig D.F. Booker S.J. Biochemistry. 2004; 43: 13496-13509Crossref PubMed Scopus (89) Google Scholar, 63Markham G.D. Hafner E.W. Tabor C.W. Tabor H. J. Biol. Chem. 1980; 255: 9082-9092Abstract Full Text PDF PubMed Google Scholar, 64Park J. Tai J.Z. Roessner C.A. Scott A.I. Bioorg. Med. Chem. Lett. 1995; 5: 2203-2206Crossref Scopus (11) Google Scholar) (for experimental details, please see "Experimental Procedures"). We recorded two-dimensional HSQC spectra of [methyl-13C]AdoMet in the presence of stoichiometric quantities of the unlabeled catalytic domain of SET7/9 (Fig. 1). The 1H chemical shift of the AdoMet methyl group was recorded as 3.8 ppm. This chemical shift was unusually far downfield for a methyl group and also represented a large downfield change relative to the reported chemical shift for the AdoMet methyl group free in solution (3.0 ppm) (65Seeger K. Lein S. Reuter G. Berger S. Biochemistry. 2005; 44: 6208-6213Crossref PubMed Scopus (9) Google Scholar). Unlike 13C, downfield 1H chemical shift changes often qualitatively indicate hydrogen bond formation (23Ash E.L. Sudmeier J.L. Day R.M. Vincent M. Torchilin E.V. Haddad K.C. Bradshaw E.M. Sanford D.G. Bachovchin W.W. Proc. Natl. Acad. Sci. 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Chem. 2005; 70: 92-100Crossref PubMed Scopus (35) Google Scholar, 71Afonin A.V. Ushakov I.A. Vashchenko A.V. Simonenko D.E. Ivanov A.V. Vasil'tsov A.M. Mikhaleva A.I. Trofimov B.A. Magn. Reson. Chem. 2009; 47: 105-112Crossref PubMed Scopus (39) Google Scholar) and in computational biology (68Scheiner S. Gu Y. Kar T. J. Mol. Struct. 2000; 500: 441-452Crossref Scopus (113) Google Scholar), it has, to our knowledge, not yet been applied experimentally in biological macromolecules. Using this combination of techniques, we reasoned that it should be possible to solve for the hydrogen positions and, thus hydrogen bonding patterns, of the AdoMet methyl group within the SET7/9 active site.Previous studies of CH···O hydrogen bonding in small organic molecules showed that the chemical shift calculations of hydrogen were usually accurate to 0.1 ppm of the experiment (37Yates J.R. Pham T.N. Pickard C.J. Mauri F. Amado A.M. Gil A.M. Brown S.P. J. Am. Chem. Soc. 2005; 127: 10216-10220Crossref PubMed Scopus (171) Google Scholar, 70Sigalov M. Vashchenko A. Khodorkovsky V. J. Org. Chem. 2005; 70: 92-100Crossref PubMed Scopus (35) Google Scholar, 71Afonin A.V. Ushakov I.A. Vashchenko A.V. Simonenko D.E. Ivanov A.V. Vasil'tsov A.M. Mikhaleva A.I. Trofimov B.A. Magn. Reson. Chem. 2009; 47: 105-112Crossref PubMed Scopus (39) Google Scholar). Error in biological molecules could arise from many sources, including but not limited to implicit solvation modeling and large or truncated molecules used in calculations. Therefore, to validate the accuracy of our calculations for the AdoMet methyl group, we first calculated the chemical shift of the AdoMet methyl group using the solution state NMR structure of free AdoMet in water. Methyl proton geometry was optimized, allowing for the appropriate number of hydrogen bonds formed in solution by the methyl group, followed by chemical shift calculation (see "Experimental Procedures" for computational details and supplemental Table S1 for a list of all chemical shifts). By averaging all three values together to reproduce the single experimental methyl resonance, the difference in chemical shift between the experimental (3.0 ppm) (65Seeger K. Lein S. Reuter G. Berger S. Biochemistry. 2005; 44: 6208-6213Crossref PubMed Scopus (9) Google Scholar) and calculated values was <0.1 ppm. This level of accuracy prompted us to attempt to locate the methyl protons of AdoMet within the active site of SET7/9.To compare with our experimental data, we then modeled the active site of SET7/9 using its crystal structure bound to AdoMet at 1.7 Å resolution (4Kwon T. Chang J.H. Kwak E. Lee C.W. Joachimiak A. Kim Y.C. Lee J. Cho Y. EMBO J. 2003; 22: 292-303Crossref PubMed Scopus (105) Google Scholar). Hydrogens were added to this model, and the geometry of the hydrogen positions was optimized (see "Experimental Procedures" for details). Chemical shifts were then calculated for the methyl group protons and averaged to a single value, as performed for free AdoMet. The geometry-optimized structure of the AdoMet methyl group shows CH···O hydrogen bond formation from the AdoMet methyl group to the hydroxyl group of Tyr-335 and the main chain carbonyl oxygen of His-293, with H···O distances of 2.5 and 2.1 Å, respectively (Fig. 2A). Moreover, the C-H···O angles of 146° and 140°, respectively, are acceptable hydrogen bonding angles (16Desiraju G.R. Acc. Chem. Res. 1991; 24: 290-296Crossref Scopus (1333) Google Scholar, 17Derewenda Z. Lee L. Kobos P. Derewenda U. FASEB J. 1995; 9: A1246Google Scholar, 72Sarkhel S. Desiraju G.R. Proteins. 2004; 54: 247-259Crossref PubMed Scopus (242) Google Scholar). As predicted, the calculations showed that protons participating in CH···O hydrogen bonds experienced significant downfield changes in chemical shift. The proton engaged in close hydrogen bonding with His-293 had a calculated chemical shift of 5.0 ppm, whereas the proton forming hydrogen bonds with Tyr-335 had a calculated chemical shift of 3.3 ppm. The chemical shift of the third proton was calculated to be 2.6 ppm. Averaging all three values together yielded a calculated chemical shift of 3.7 ppm, which is within 0.1 ppm of the experimental value of 3.8 ppm.FIGURE 2Optimized active site with bound AdoMet (A) and manually rotated geometry (B) to eliminate CH···O hydrogen bonds. Truncated AdoMet and the protein are depicted with green and gray carbon atoms, respectively. Residues labeled in red designate CH···O acceptors. H···O distances from methyl protons to nearest oxygen atom for optimized and broken geometry are shown in magenta and cyan, respectively. The crystal structure of the SET7/9·AdoMet complex (Protein Data Bank code 1N6A) was used as the model for the calculations.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To further confirm the presence of CH···O hydrogen bonding, the methyl group was rotated manually through a range of 120° (example shown in Fig. 2B), and chemical shifts were recalculated at intermediate geometries to explore whether alternative hydrogen positions could agree with the experimental data. The rotation of the AdoMet methyl group resulted in a smooth functional change in the calculated chemical shift, passing through a minimum at 36° (Fig. 3). The minimum of this plot corresponded to the structure for which the model and the experimentally measured chemical shift differed by 0.6 ppm. This difference represents the following: 1) the largest deviation between the experiment and model, 2) the most upfield of the calculated chemical shifts, and 3) the model in which the methyl rotamer forms minimal CH···O hydrogen bonds in the active site (Fig. 2B). These calculations indicated that AdoMet methyl rotamers precluding CH···O hydrogen bond formation were inconsistent with the experimental chemical shift data. All of the calculated chemical shifts that agreed closely with experimental chemical shifts represented structures that optimized CH···O hydrogen bonding for at least one AdoMet methyl hydrogen atom. Collectively, the experimental data and calculations yielded direct evidence via NMR chemical shift for CH···O hydrogen bond formation between the AdoMet methyl group and oxygen atoms within the active site of SET7/9.FIGURE 3Chemical shift of the AdoMet methyl group as a function of rotation angle. 0° and 120°, rendered in green, are equivalent geometry optimized positions (displayed in Fig. 2A), whereas all other points were derived from manual rotation of the AdoMet methyl group. The point at 36° represents the chemical shift calculated from the AdoMet methyl conformation shown in Fig. 2B. The blue and red dashed lines indicate the experimental chemical shift and the structure calculated to contain the least CH···O hydrogen bonding, respectively.View Large Image Figure ViewerDownload Hi-res image Download (PPT)These findings prompted us to examine the thermodynamic parameters for cofactor binding by SET7/9 using ITC (see "Experimental Procedures" for experimental details). Binding affinities and enthalpies for SET7/9 were measured with AdoMet (Fig. 4), its methyl transfer product AdoHcy, and sinefungin, an AdoMet analogue in which the methyl sulfonium cation is substituted by an amine-methylene group that can participate in conventional NH···O hydrogen bonding. Crystal structures of SET7/9 and other SET domain enzymes illustrated that these cofactors share a structurally homologous binding mode (supplemental Fig. S4) (4Kwon T. Chang J.H. Kwak E. Lee C.W. Joachimiak A. Kim Y.C. Lee J. Cho Y. EMBO J. 2003; 22: 292-303Crossref PubMed Scopus (105) Google Scholar, 5Xiao B. Jing C. Wilson J.R. Walker P.A. Vasisht N. Kelly G. Howell S. Taylor I.A. Blackburn G.M. Gamblin S.J. Nature. 2003; 421: 652-656Crossref PubMed Scopus (302) Google Scholar, 7Subramanian K. Jia D. Kapoor-Vazirani P. Powell D.R. Collins R.E. Sharma D. Peng J. Cheng X. Vertino P.M. Mol. Cell. 2008; 30: 336-347Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar). The ITC data revealed that SET7/9 displayed nanomolar affinities for AdoMet and sinefungin, whereas its affinity for AdoHcy was ∼1000-fold weaker (Table 1). These data were analogous to those obtained for the binding of these ligands to the SET domain protein Rubisco large subunit methyltransferase (LSMT), demonstrating consistency in rank order of binding affinities across different SET domain enzymes (6Couture J.F. Hauk G. Thompson M.J. Blackburn G.M. Trievel R.C. J. Biol. Chem. 2006; 281: 19280-19287Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). In addition, SET7/9 and LSMT displayed comparable differences in binding enthalpy between AdoMet and sinefungin (∼3 kcal/mol). The high affinity that these enzymes displayed for AdoMet is presumably due to the ability of its methyl group to engage in CH···O hydrogen bonding, emphasizing the importance of these interactions in cofactor binding by SET domain KMTs.FIGURE 4ITC analysis of AdoMet binding to SET7/9. The top panel represents the titration of AdoMet into SET7/9, whereas the bottom panel represents the binding isotherm with fitted curve.View Large Image Figure ViewerDownload Hi-res image Download (PPT)DISCUSSIONThe identification of CH···O hydrogen bonding between the AdoMet methyl group and oxygen atoms within the SET domain active site has implications for these interactions in lysine methyl transfer reactions. As proposed previously, CH···O hydrogen bonds appear to play roles in binding AdoMet, positioning its methyl group in an appropriate geometry for transfer, and stabilizing the SN2 transition state (6Couture J.F. Hauk G. Thompson M.J. Blackburn G.M. Trievel R.C. J. Biol. Chem. 2006; 281: 19280-19287Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). These data suggest that the CH···O hydrogen bonds confer a specific orientation for the methyl group to align it during catalysis and could potentially limit its motion within the active site. Future studies will further define the specific roles that CH···O hydrogen bonds may play in promoting the methyl transfer reaction catalyzed by SET domain enzymes.The thermodynamic analyses of SET7/9 bound to AdoMet and its analogues are consistent with previous findings on LSMT (6Couture J.F. Hauk G. Thompson M.J. Blackburn G.M. Trievel R.C. J. Biol. Chem. 2006; 281: 19280-19287Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). The consistency of rank order in binding affinity indicates that CH···O hydrogen bonds are important in cofactor binding by multiple KMTs due to the structural conservation of the SET domain active site. Moreover, these CH···O interactions may in part explain the importance of the evolutionarily invariant Tyr-335 to enzyme function (Fig. 2A), given the propensity of its hydroxyl group to form CH···O hydrogen bonds with the AdoMet methyl group. It is also interesting to note that the change in binding affinity between AdoMet and its analogues is substantially more dramatic in SET7/9 than in LSMT. This effect could be a function of the plasticity of the SET7/9 active site (9Xiao B. Jing C. Kelly G. Walker P.A. Muskett F.W. Frenkiel T.A. Martin S.R. Sarma K. Reinberg D. Gamblin S.J. Wilson J.R. Gene Dev. 2005; 19: 1444-1454Crossref PubMed Scopus (152) Google Scholar), as compared with the preformed active site of LSMT (42Trievel R.C. Flynn E.M. Houtz R.L. Hurley J.H. Nat. Struct. Biol. 2003; 10: 545-552Crossref PubMed Scopus (99) Google Scholar). Future studies may address how conformational flexibility within the SET domain family influences CH···O hydrogen bonding to AdoMet.In terms of methodology, our results have demonstrated that chemical shift can be used as a structural parameter for determining hydrogen positions and hydrogen bonding patterns within an enzyme active site. To our knowledge, these chemical shift data and calculations provide the first direct, quantitative evidence of CH···O hydrogen bonding in an enzyme active site in solution. One advantage of chemical shift as a probe to examine CH···O hydrogen bonding in biomolecular structure is the relative ease of data acquisition. In the future, this methodology could be applied broadly to characterize CH···O hydrogen bonding in proteins, nucleic acids, and other biological molecules, expanding our understanding of the functional importance of these interactions in macromolecular structure, ligand binding, and enzyme catalysis. IntroductionPost-translational modifications in proteins are now well recognized as important players in many biological processes. Among these modifications, site-specific lysine methylation by SET domain KMTs 3The abbreviations used are: KMT, lysine methyltransferase; AdoMet, S-adenosylmethionine; CH···O, carbon-oxygen hydrogen bond; B3LYP, Becke, three-parameter, Lee-Yang-Parr; MD, molecular dynamics; HSQC, heteronuclear single quantum coherence; ITC, isothermal titration calorimetry; AdoHcy, S-adenosylhomocysteine; LSMT, Rubisco large subunit methyltransferase. is known to be critical to a diverse set of processes within the nucleus, including gene expression, cell cycle progression, and DNA damage response (1Albert M. Helin K. Semin. Cell Dev. Biol. 2010; 21: 209-220Crossref PubMed Scopus (228) Google Scholar, 2Dillon S.C. Zhang X. Trievel R.C. Cheng X.D. Genome Biol. 2005; 6: 227Crossref PubMed Scopus (547) Google Scholar). 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Chem. 2006; 281: 19280-19287Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 38Couture J.F. Dirk L.M. Brunzelle J.S. Houtz R.L. Trievel R.C. Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 20659-20664Crossref PubMed Scopus (89) Google Scholar, 39Del Rizzo P.A. Couture J.F. Dirk L.M. Strunk B.S. Roiko M.S. Brunzelle J.S. Houtz R.L. Trievel R.C. J. Biol. Chem. 2010; 285: 31849-31858Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). These interactions were identified in x-ray crystal structures, in which C···O distances between the AdoMet methyl carbon, structurally conserved carbonyl oxygen atoms, and an invariant tyrosine were appropriate (<3.7 Å) for CH···O hydrogen bonding to occur (6Couture J.F. Hauk G. Thompson M.J. Blackburn G.M. Trievel R.C. J. Biol. Chem. 2006; 281: 19280-19287Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar) (supplemental Fig. S1). These hydrogen bonds can form because the electron withdrawing character of the sulfonium cation polarizes the methyl group of AdoMet. Isothermal calorimetry demonstrated that AdoMet displays higher binding affinity to a SET domain KMT than AdoMet analogues that are incapable of forming CH···O hydrogen bonds (6Couture J.F. Hauk G. Thompson M.J. Blackburn G.M. Trievel R.C. J. Biol. Chem. 2006; 281: 19280-19287Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar), indicating the importance of these interactions in substrate binding. In addition, it was postulated that the CH···O hydrogen bonds align the AdoMet methyl group in the requisite linear geometry with the substrate lysine ϵ-amine group for the SN2 methyl transfer reaction. (supplemental Fig. S2) (2Dillon S.C. Zhang X. Trievel R.C. Cheng X.D. Genome Biol. 2005; 6: 227Crossref PubMed Scopus (547) Google Scholar, 5Xiao B. Jing C. Wilson J.R. Walker P.A. Vasisht N. Kelly G. Howell S. Taylor I.A. Blackburn G.M. Gamblin S.J. Nature. 2003; 421: 652-656Crossref PubMed Scopus (302) Google Scholar, 6Couture J.F. Hauk G. Thompson M.J. Blackburn G.M. Trievel R.C. J. Biol. Chem. 2006; 281: 19280-19287Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 40Trievel R.C. Beach B.M. Dirk L.M. Houtz R.L. Hurley J.H. Cell. 2002; 111: 91-103Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar, 41Zhang X. Yang Z. Khan S.I. Horton J.R. Tamaru H. Selker E.U. Cheng X.D. Mol. Cell. 2003; 12: 177-185Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar). However, as hydrogen atoms are not observed in all but the highest resolution x-ray crystal structures, it is possible that the AdoMet methyl protons occupy positions that preclude CH···O hydrogen bond formation, despite close C···O contacts within SET domain crystal structures. Thus, the presence of CH···O hydrogen bonding can only be inferred from these structures. Here, we address the fundamental question, is there direct experimental evidence for CH···O hydrogen bonding between the AdoMet methyl group and the SET domain active site in solution?
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