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

Joan M. Hevel,Shanying Gui,Paula J. Porter,Sean J. Johnson,Michael P. Daly,Whitney L. Wooderchak

doi:10.1074/jbc.m111.224097

Joan M. Hevel, Shanying Gui + Show 4 more

Open Access

https://doi.org/10.1074/jbc.m111.224097

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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.

Highlights

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
It has been noted that all the type I PRMTs contain an active site methionine, which has been hypothesized to dictate the synthesis of asymmetric dimethyl arginine (ADMA) over symmetric dimethyl arginine (SDMA) by preventing the binding of monomethyl arginine (MMA) in a configuration conducive for SDMA formation [12, 13]
Given the potential for either methionine to influence substrate geometry in the active site, we investigated whether these residues are involved in specifying ADMA formation versus SDMA

Summary

Introduction

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. A key to understanding the biological function of the PRMTs is to understand how product specificity may be regulated, in terms of governing which arginyl residues are modified and which state of methylation is achieved. Structural studies of PRMT1 [11] and PRMT3 [12], both type I methyltransferases, have identified the component residues of the type I active site and have provided a basis from which to probe product specificity. It has been noted that all the type I PRMTs contain an active site methionine (position 155 in rat PRMT1), which has been hypothesized to dictate the synthesis of ADMA over SDMA by preventing the binding of MMA in a configuration conducive for SDMA formation [12, 13]. Our data show that Met-48 plays a crucial role in specifying which peptidyl arginine is targeted by PRMT1

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