Investigation of the Molecular Origins of Protein-arginine Methyltransferase I (PRMT1) Product Specificity Reveals a Role for Two Conserved Methionine Residues

Abstract

Protein-arginine methyltransferases aid in the regulation of many biological processes by methylating specific arginyl groups within targeted proteins. The varied nature of the response to methylation is due in part to the diverse product specificity displayed by the protein-arginine methyltransferases. In addition to site location within a protein, biological response is also determined by the degree (mono-/dimethylation) and type of arginine dimethylation (asymmetric/symmetric). Here, we have identified two strictly conserved methionine residues in the PRMT1 active site that are not only important for activity but also control substrate specificity. Mutation of Met-155 or Met-48 results in a loss in activity and a change in distribution of mono-and dimethylated products. The altered substrate specificity of M155A and M48L mutants is also evidenced by automethylation. Investigation into the mechanistic basis of altered substrate recognition led us to consider each methyl transfer step separately. Single turnover experiments reveal that the rate of transfer of the second methyl group is much slower than transfer of the first methyl group in M48L, especially for arginine residues located in the center of the peptide substrate where turnover of the monomethylated species is negligible. Thus, altered product specificity in M48L originates from the differential effect of the mutation on the two rates. Characterization of the two active-site methionines provides the first insight into how the PRMT1 active site is engineered to control product specificity.Protein methylation is a significant post-translational modification in eukaryotic organisms. Protein arginine residues can be methylated on the guanidino nitrogens by protein-arginine methyltransferases (PRMTs), 2 which use S-adenosyl-L-methionine (AdoMet) as a methyl group donor. This post-translational modification is important in a wide variety of fundamental biological processes, including transcription, RNA splicing, signal transduction, DNA repair, viral replication (reviewed in Ref. 1), and chromatin remodeling (2). In recent years, the significance of PRMTs in human diseases has been increasingly studied, especially in cardiovascular disease (3) and cancer (4). In all, PRMTs play a crucial role in many biological processes.Although the biological importance of PRMTs has become well accepted, the current knowledge of the fundamental biochemistry of these enzymes is limited, due in part to the complexity of the system. So far, 11 PRMT isoforms have been identified. In mammalian cells, nine PRMTs catalyze monomethyl arginine (MMA) formation, and they can be categorized into two major types as follows: PRMT1, -2-4, -6, and -8 additionally catalyze asymmetric dimethyl arginine (ADMA) formation, demonstrating type I activity; and PRMT5, -7, and -9 catalyze symmetric dimethyl arginine (SDMA) formation, demonstrating type II activity (Fig. 1A). PRMT10 and -11 were identified as putative PRMT genes with no methylation activity shown as yet (5). As with other enzyme f...

QM/MM MD and free energy simulations of the methylation reactions catalyzed by protein arginine methyltransferase PRMT3

“…Several experimental studies have been performed to address this question. [27][28][29][30] For Type I enzymes, it was proposed that the active site methionine residues might prevent the binding of -MMA in a conformation that allows the formation of symmetric product. For Type II enzymes, this position is occupied by the residues with smaller side chains, such as Ser as observed in PRMT5.…”

“…This seems to suggest that these methionines may not be responsible for the failure of the formation of SDMA in the Type I enzymes. 28,29 In this paper, QM/MM MD and free energy simulations were performed to investigate the energetic origin of the product specificity of PRMT3, a Type I PRMT that generate ADMA instead of SDMA. It has been discussed in our previous studies of PKMTs that the product specificity of PKMTs could be reflected from MD and free energy simulations.…”

A Remodeled Protein Arginine Methyltransferase 1 (PRMT1) Generates Symmetric Dimethylarginine

“…Guided by the crystal structure of PRMT1 (12), the active site of PRMT1 has been dissected to understand the kinetic mechanism (13)(14)(15) and product specificity (16). In our recent work, we identified that two conserved active site residues, Met-48 and Met-155, play a significant role in enzymatic activity and regulation of mono-versus asymmetric dimethylation (16).…”

“…In our recent work, we identified that two conserved active site residues, Met-48 and Met-155, play a significant role in enzymatic activity and regulation of mono-versus asymmetric dimethylation (16). Although Met-155 was suggested to govern formation of ADMA over SDMA (12,17), mutating either Met-155 or Met-48 to small amino acids, such as Ala or Leu, did not change the dimethylation product (14,16). When the structure of PRMT5 was solved, a conserved phenylalanine, Phe-379, was surprisingly found at the corresponding location in the active site as Met-48 in rat PRMT1 (18).…”

Methylation of the nuclear poly(A)-binding protein by type I protein arginine methyltransferases – how and why

“…Evidence has been presented that, in contrast to earlier proposals, the deprotonation of the guanidinium group is not an essential part of the mechanism and methyl transfer may be promoted predominantly by the proximity between the guanidinium group and the methyl group of SAM in the active site (Rust et al , 2011 ). A pair of methionine residues has been proposed to be important in orienting the arginine side chain (Gui et al , 2011 ).…”