MT-MAMS: Protein Methyltransferase Motif Analysis by Mass Spectrometry

Abstract

Protein methyltransferases often recognize their substrates through linear sequence motifs. The determination of these motifs is critical to understand methyltransferase mechanism, function, and drug targeting. Here we describe MT-MAMS (methyltransferase motif analysis by mass spectrometry), a quantitative approach to characterize methyltransferase substrate recognition motifs. In MT-MAMS, peptide sets are synthesized which contain all amino acid substitutions at single positions within a template sequence. These are then incubated with the methyltransferase of interest in the presence of deuterated S-adenosyl methionine (D-AdoMet). The use of this heavy methyl donor gives unique mass shifts to methylated peptides, allowing their unambiguous quantification by mass spectrometry. The stoichiometry of methylation resulting from each substitution is then derived, and finally the methyltransferase substrate recognition motif is generated. We validated MT-MAMS by application to lysine methyltransferase G9a, generating the substrate recognition motif (TKRN)-(A > RS > G)-(R ≫ K)-K-(STRCKMAQHG)-Φ; this is highly similar to that previously determined by peptide arrays. We then determined the recognition motif of yeast lysine elongation factor methyltransferase 1 (Efm1) to be (Y > FW)-K-^P-G-G-Φ. This is a new type of lysine methyltransferase recognition motif that only contains noncharged residues, excluding the target lysine. We further determined recognition motifs of major yeast and human arginine methyltransferases Hmt1 and PRMT1, revealing them to be ^(DE)-^(DE)-R-(G ≫ A)-(GN > RAW)-(FYW > ILKHM) and ^(DE)-^(DE)-R-(G ≫ N)-(GR > ANK)-(K > YHMFILW), respectively. These motifs expand significantly on the canonical RGG recognition motif and include the negative specificity of these enzymes, a feature unique to MT-MAMS. Finally, we show that MT-MAMS can be used to generate insights into the processivity of protein methyltransferases.

Phosphorylation Regulates CIRBP Arginine Methylation, Transportin-1 Binding and Liquid-Liquid Phase Separation

“…Thus, we suggest the vicinity of negatively charged phosphate groups to target arginines prevents binding to PRMT1 active site and methylation of arginines 94, 112, and 116 due to the electrostatic repulsion. This is in line with the observation that negatively charged amino acids next to the arginine disfavour methylation ( Hamey et al, 2018 ). Hence, our findings indicate that arginine methylation and serine phosphorylation of CIRBP-RGG directly modulate each other ( Figure 7 ).…”

Protein Methylation and Translation: Role of Lysine Modification on the Function of Yeast Elongation Factor 1A

“…Recently, evidence of the in vitro methylation of an EF1A-derived peptide containing Lys-253 in S. cerevisiae by Efm1 was presented. 59 This lysine residue is found in a sequence motif similar to that of the Efm1 Lys-30 site. It is possible that methylation at Lys-253 could be contributing to the monomethylation peak observed in the cation exchange chromatography of the TEF1 K(30,79,316,390)R mutant.…”

Using Yeast to Define the Regulatory Role of Protein Lysine Methylation

“…It is possible that both Efm1 and Efm4/See1 separately target eEF1A K30, or they potentially interact with each other or function together to catalyze this methylation. Although Efm4/See1 is able to directly methylate eEF1A at K316 in vitro, it does not methylate K30 in vitro, whereas Efm1 is able to methylate K30 and K253 in vitro [21,38]. Further biochemical investigation may reveal whether these proteins are associated with complexes or other co-factors that direct methylation of eEF1A K30 in cells.…”