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

Abstract

SET domain lysine methyltransferases (KMTs) are S-adenosylmethionine (AdoMet)-dependent enzymes that catalyze the site-specific methylation of lysyl residues in histone and nonhistone proteins. Based on crystallographic and cofactor binding studies, carbon-oxygen (CH⅐⅐⅐O) 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 CH⅐⅐⅐O hydrogen bonding. These findings represent direct, quantitative evidence of CH⅐⅐⅐O 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.Post-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 3 is known to be critical to a diverse set of processes within the nucleus, including gene expression, cell cycle progression, and DNA damage response (1, 2). 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 (3). 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 (4 -13). Despite these studies, many aspects regarding its methyl transfer reaction mechanism remain unclear, including the possibility that unconventional CH⅐⅐⅐O hydrogen bonds participate in catalysis (6).CH⅐⅐⅐O hydrogen bonding has been recognized as an important interaction in proteins and other biological macromolecules dating back 40 years (14 -17). For example, it has been estimated that 17% of the energy percentage at protein-protein surfaces is due to CH⅐⅐⅐O hydrogen bonding, and at some protein surfaces, that percentage is as high as 40 -50% (18). These hydrogen bonds also have been implicated in enzyme catalysis (19 -23), stabilizing nucleic acid structure (24 -27), as well as interactions with methyl groups in small molecules (28 -32). Despite their importance, experimental characterization of CH⅐⅐⅐O hydrogen bonds in proteins remains challenging. Current methods for identifying are difficult to employ for many proteins, including SET7/9. However, NMR spectroscopy holds promise in identifying CH⅐⅐⅐O hydrogen b...

Mediation of donor–acceptor distance in an enzymatic methyl transfer reaction

“…One possible explanation for BIEs >1 is a change in local electrostatics that fails to be accompanied by any overall decrease in the distance between the methyl donor (S + ) and acceptor (represented by the hydroxyl group of 8-hydroxyquinoline). We note that in a related methyl transfer system, SET7/9, X-ray crystallography and NMR of the binary complex have led to the proposal of ground state hydrogen bonding between the C-H of the methyl group in AdoMet and electronaccepting protein side chain(s) (45,46). A role for such putative C-H···O bonding in reducing vibrational frequencies at the transferred methyl group would be consistent with BIEs larger than unity, with the caveat that the elevated BIE data presented herein are for a different enzyme family and only occur in the presence of the relatively weak binding inhibitor 8-hydroxyquinoline.…”

Kinetic isotope effects reveal early transition state of protein lysine methyltransferase SET8

“…However, limited efforts have been made to explore experimentally the (S N 2) TS of PKMT-catalyzed methylation. Recent evidence from static PKMT structures and dynamic NMR chemical shifts suggests the existence of equatorial noncanonical CH-O interaction between the sulfonium methyl moiety of SAM and the oxygens of active-site amino acid residues of PKMTs (36)(37)(38), although it remains to examine how the equatorial CH-O interaction participates in the formation of the TS of PKMT-catalyzed methylation reaction.…”

“…The recent elucidation of noncanonical CH-O interaction could shed light on an alternative, but mutually nonexclusive mechanism for the constrained TS of protein methyltransferases (36)(37)(38). In the context of the TS of SET8-catalyzed H4K20 monomethylation, the sulfonium-methyl group of SAM is expected to form three equatorial noncanonical CH-O interactions with the two backbone amide oxygens of Cys270 and Arg295 and the phenolic oxygen of the side chain of Tyr336 (Fig.…”

H3K36 Methyltransferases as Cancer Drug Targets: Rationale and Perspectives for Inhibitor Development

“…The methyl donor SAM binds in a pocket at the junction of core SET, SET-I and post-SET, which make extensive contacts to SAM including hydrogen bonds and van der Waals contacts. Other important interactions involve tyrosine and carbonyl oxygens that form CH···O hydrogen bonds with the SAM methyl group [126].…”

“…The methyl donor SAM binds in a pocket at the junction of core SET, SET-I and post-SET, which make extensive contacts to SAM including hydrogen bonds and van der Waals contacts. Other important interactions involve tyrosine and carbonyl oxygens that form CH···O hydrogen bonds with the SAM methyl group [126].The autoinhibitory loop must undergo a significant conformational change to accommodate nucleosome substrate, and interestingly this loop undergoes dynamics even without nucleosome present. Molecular dynamics simulations, crystallographic and NMR studies have shown that the NSD1 and ASH1L autoinhibitory loops experience conformational heterogeneity in the absence of nucleosome substrate [127,128].…”