Abstract
In the family of protein arginine methyltransferases (PRMTs) that predominantly generate either asymmetric or symmetric dimethylarginine (SDMA), PRMT7 is unique in producing solely monomethylarginine (MMA) products. The type of methylation on histones and other proteins dictates changes in gene expression, and numerous studies have linked altered profiles of methyl marks with disease phenotypes. Given the importance of specific inhibitor development, it is crucial to understand the mechanisms by which PRMT product specificity is conferred. We have focused our attention on active-site residues of PRMT7 from the protozoan Trypanosoma brucei We have designed 26 single and double mutations in the active site, including residues in the Glu-Xaa8-Glu (double E) loop and the Met-Gln-Trp sequence of the canonical Thr-His-Trp (THW) loop known to interact with the methyl-accepting substrate arginine. Analysis of the reaction products by high resolution cation exchange chromatography combined with the knowledge of PRMT crystal structures suggests a model where the size of two distinct subregions in the active site determines PRMT7 product specificity. A dual mutation of Glu-181 to Asp in the double E loop and Gln-329 to Ala in the canonical THW loop enables the enzyme to produce SDMA. Consistent with our model, the mutation of Cys-431 to His in the THW loop of human PRMT9 shifts its product specificity from SDMA toward MMA. Together with previous results, these findings provide a structural basis and a general model for product specificity in PRMTs, which will be useful for the rational design of specific PRMT inhibitors.
Highlights
Methylation of proteins is a major type of post-translational modification involved in the regulation of a variety of cellular processes mediated by protein-protein interactions, including splicing, transcription, translation, and signaling (1–3)
These enzymes can be further categorized based on which methylarginine product they catalyze as follows: type I protein arginine methyltransferases (PRMTs) catalyze the production of -NG-monomethylarginine (MMA) and asymmetric -NG,NG-dimethylarginine (ADMA); type II PRMTs catalyze the production of MMA and symmetric -NG,NЈGdimethylarginine (SDMA); type III PRMTs catalyze the production of only MMA; and type IV PRMTs catalyze ␦NG-monomethylarginine production (11)
TbPRMT7 Active-site Double Mutation, E181D/Q329A, Converts the Enzyme to an SDMA-producing PRMT—Given the ability of the double E loop E181D mutation of TbPRMT7 to alter the methylation type (25), seven TbPRMT7 double mutants were generated with the E181D background to probe the effects of further increasing the size of the active site
Summary
PRMT5 (24), but they have not put forth a general model for the factors that guide product specificity for the three main types of PRMTs. Using an approach where MMA, ADMA, and SDMA can be detected with sub-femtomole sensitivity, we have been able to demonstrate the transformation of PRMT7 from Trypanosoma brucei (TbPRMT7) from an enzyme that strictly produces MMA to one forming ADMA by replacing a glutamate residue in the double E loop (Glu-181) with an aspartate residue (Fig. 1) (25). We have focused on TbPRMT7 because it displays robust type III activity and has been amenable to structural analysis (25–27). Complementary studies with PRMT9 from Homo sapiens, previously characterized as a type II enzyme (28, 29), corroborate our PRMT7 results. Based on this evidence, we propose a structural model for how PRMTs can limit their activities to type I, type II, or type III methylation
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