Abstract
SET domain enzymes represent a distinct family of protein lysine methyltransferases in eukaryotes. Recent studies have yielded significant insights into the structural basis of substrate recognition and the product specificities of these enzymes. However, the mechanism by which SET domain methyltransferases catalyze the transfer of the methyl group from S-adenosyl-L-methionine to the lysine epsilon-amine has remained unresolved. To elucidate this mechanism, we have determined the structures of the plant SET domain enzyme, pea ribulose-1,5 bisphosphate carboxylase/oxygenase large subunit methyltransferase, bound to S-adenosyl-L-methionine, and its non-reactive analogs Aza-adenosyl-L-methionine and Sinefungin, and characterized the binding of these ligands to a homolog of the enzyme. The structural and biochemical data collectively reveal that S-adenosyl-L-methionine is selectively recognized through carbon-oxygen hydrogen bonds between the cofactor's methyl group and an array of structurally conserved oxygens that comprise the methyl transfer pore in the active site. Furthermore, the structure of the enzyme co-crystallized with the product epsilon-N-trimethyllysine reveals a trigonal array of carbon-oxygen interactions between the epsilon-ammonium methyl groups and the oxygens in the pore. Taken together, these results establish a central role for carbon-oxygen hydrogen bonding in aligning the cofactor's methyl group for transfer to the lysine epsilon-amine and in coordinating the methyl groups after transfer to facilitate multiple rounds of lysine methylation.
Highlights
Protein lysine methylation has emerged as a prominent post-translational modification in gene regulatory and intracellular signaling pathways
Based on the structure of the pea Rubisco large subunit methyltransferase (pLSMT)-MeLys complex, we proposed that CH1⁄71⁄71⁄7O hydrogen bonding between the methyl transfer pore and mono- and dimethyllysine facilitates subsequent rounds of methylation by orienting the ⑀-amine toward the methyl transfer pore while rotating the existing methyl groups out of the transfer path [13]
Calorimetric Analysis of AdoMet, AdoHcy, AzaAdoMet, and Sinefungin Binding to aLSMT—To investigate the interactions of different chemical moieties within the methyl transfer pore of pLSMT, we determined the thermodynamic parameters for the binding of AdoMet, Sinefungin, and AzaAdoMet to a homolog of the enzyme using isothermal titration calorimetry (ITC)
Summary
Chemicals and Reagents—S-Adenosyl-L-methionine, S-adenosyl-L-homocysteine, L-lysine acetate, and Sinefungin were purchased from Sigma, and L-⑀-monomethyllysine and L-⑀-trimethyllysine were obtained from Bachem. AdoMet was further purified by anion-exchange chromatography as reported previously [39]. L-AzaAdoMet was synthesized and purified according to the method of Thompson et al [40]. Protein Cloning, Expression, and Purification—The DNA encoding residues 43– 477 of Arabidopsis thaliana Rubisco
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