Protein Arginine Methyltransferase Product Specificity Is Mediated by Distinct Active-site Architectures

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-Xaa 8 -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.

Caenorhabditis elegans PRMT-7 and PRMT-9 Are Evolutionarily Conserved Protein Arginine Methyltransferases with Distinct Substrate Specificities

“…Mutation of the THW loop residue A391 to the conserved histidine residue abolishes the SDMA activity, producing only MMA, consistent with previous work with the human enzyme PRMT9 C431H mutant 22 . These reactions were single replicates.…”

“…Although the characterized mammalian, trypanosome, and C. elegans PRMT7 homologs all produce MMA, there are significant differences in their substrate specificity (this study, 18,22 ). We therefore examined the active site architecture of the available PRMT7 crystal structures of each of these enzymes.…”

“…We therefore examined the active site architecture of the available PRMT7 crystal structures of each of these enzymes. The substrate arginine residue is generally bound between the two glutamate residues in the double E loop 3,4,22 . The distance between the side chain carboxyls of the two glutamates in the double E loop is relatively similar (7.6 Å and 7.2 Å) in the C. elegans and T. brucei enzymes, compared to a greater distance (8.7 Å) in the M. musculus enzyme (Fig.…”

“…Due to the complexity of mammalian systems, PRMT7 from lower eukaryotes including Trypanosoma brucei 19–22 and Caenorhabditis elegans 23,24 has been studied. However, the trypanosome enzyme has preference for RG-rich proteins such as RBP16 19,21,22 , while mammalian PRMT7 has a preference for R-X-R motifs in basic sequence contexts such as those found in histone H2B 17,18 .…”

“…However, the trypanosome enzyme has preference for RG-rich proteins such as RBP16 19,21,22 , while mammalian PRMT7 has a preference for R-X-R motifs in basic sequence contexts such as those found in histone H2B 17,18 . A crystal structure is available for C. elegans PRMT-7 that is similar to that determined for M. musculus PRMT7, with both enzymes containing a single chain with two ancestrally duplicated methyltransferase domains stabilized by a zinc ion 24,25 .…”