Abstract
Most characterized protein methylation events encompass arginine and lysine N-methylation, and only a few cases of protein methionine thiomethylation have been reported. Newly discovered oncohistone mutations include lysine-to-methionine substitutions at positions 27 and 36 of histone H3.3. In these instances, the methionine substitution localizes to the active-site pocket of the corresponding histone lysine methyltransferase, thereby inhibiting the respective transmethylation activity. SET domain-containing 3 (SETD3) is a protein (i.e. actin) histidine methyltransferase. Here, we generated an actin variant in which the histidine target of SETD3 was substituted with methionine. As for previously characterized histone SET domain proteins, the methionine substitution substantially (76-fold) increased binding affinity for SETD3 and inhibited SETD3 activity on histidine. Unexpectedly, SETD3 was active on the substituted methionine, generating S-methylmethionine in the context of actin peptide. The ternary structure of SETD3 in complex with the methionine-containing actin peptide at 1.9 Å resolution revealed that the hydrophobic thioether side chain is packed by the aromatic rings of Tyr312 and Trp273, as well as the hydrocarbon side chain of Ile310 Our results suggest that placing methionine properly in the active site-within close proximity to and in line with the incoming methyl group of SAM-would allow some SET domain proteins to selectively methylate methionine in proteins.
Highlights
Post-translational methylation on proteins, histones, plays a fundamental role in regulating gene expression and chromatin organization [1,2,3]
Structural characterization of histone MTases in complex with the methioninesubstituted histone H3 peptides, including G9a-H3K9M [10, 11], PRC2-H3K27M [12], and SETD2-H3K36M [13], revealed that the methionine residue localizes to the active-site pocket that normally accommodates the target lysine residue, thereby inhibiting the respective transmethylation activity. It was the study of LSD1 that first used a H3 peptide bearing methionine in place of methyl-Lys4 (PDB code 2V1D), which showed that the K4M peptide is a strong inhibitor of LSD1 because the methionine substitution led to a 30-fold increase in binding affinity [14]
Protein methionine methylation here we report that unexpectedly, SET domain–containing 3 (SETD3) has methylation activity on an actin variant in which the target His73 is substituted by a methionine (H73M)
Summary
Post-translational methylation on proteins, histones, plays a fundamental role in regulating gene expression and chromatin organization [1,2,3]. Structural characterization of histone MTases in complex with the methioninesubstituted histone H3 peptides, including G9a-H3K9M [10, 11], PRC2-H3K27M [12], and SETD2-H3K36M [13], revealed that the methionine residue localizes to the active-site pocket that normally accommodates the target lysine residue, thereby inhibiting the respective transmethylation activity. In vitro, it was the study of LSD1 (a histone H3K4me2/1 demethylase) that first used a H3 peptide bearing methionine in place of methyl-Lys (PDB code 2V1D), which showed that the K4M peptide is a strong inhibitor of LSD1 because the methionine substitution led to a 30-fold increase in binding affinity [14]. We investigate the structural and molecular determinants of generating S-methylmethionine in the context of a protein peptide
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