Direct Evidence for Methyl Group Coordination by Carbon-Oxygen Hydrogen Bonds in the Lysine Methyltransferase SET7/9

Joseph D. Yesselman,Hashim M. Al-Hashimi,Scott Horowitz,Raymond C. Trievel

doi:10.1074/jbc.m111.232876

Joseph D. Yesselman, Hashim M. Al-Hashimi

Open Access

https://doi.org/10.1074/jbc.m111.232876

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Abstract

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 (1)H 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.

Highlights

Ular, the human KMT SET7/9 has been shown to methylate lysine residues on many histone and non-histone proteins and is considered to be important in many cellular pathways [3]
To probe CH1⁄71⁄71⁄7O 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
CH1⁄71⁄71⁄7O 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 [6]. These data suggest that the CH1⁄71⁄71⁄7O 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

Summary

EXPERIMENTAL PROCEDURES

Expression and Purification of MetK and SET7/9—A plasmid encoding the AdoMet synthetase (MetK) gene from Methanococcus jannaschii was generously provided by George D. To correctly model the solution state of the AdoMet methyl group, one weak CH1⁄71⁄71⁄7O hydrogen bond was included in the chemical shift calculations. The geometry of this interaction (C-H1⁄71⁄71⁄7O angle ϭ 131° and H1⁄71⁄71⁄7O interaction distance ϭ 2.5 Å) was consistent with a weak hydrogen bond This interaction satisfied our condition to include one weak CH1⁄71⁄71⁄7O hydrogen bond in the quantum chemistry calculations to correctly represent AdoMet in water, as determined by the MD simulation described above. All added hydrogen positions were optimized using B3LYP/3–21G* [59, 60], and methyl protons were subsequently optimized, and chemical shifts were calculated with B3LYP/6 –311ϩG(2d,p) as described for free AdoMet. Using implicit solvent with a lower dielectric constant to reflect the hydrophobic core of proteins (⑀ ϭ 4.9) for chemical shift calculations yielded no change in the 1H chemical shift of the AdoMet methyl group as compared with implicit water solvation. Dissociation constants and binding enthalpies of AdoMet, sinefungin, and AdoHcy to SET7/9

AdoMet Sinefungin AdoHcy nM

RESULTS

DISCUSSION