A glutamate/aspartate switch controls product specificity in a protein arginine methyltransferase

Abstract

Trypanosoma brucei PRMT7 (TbPRMT7) is a protein arginine methyltransferase (PRMT) that strictly monomethylates various substrates, thus classifying it as a type III PRMT. However, the molecular basis of its unique product specificity has remained elusive. Here, we present the structure of TbPRMT7 in complex with its cofactor product S-adenosyl-L-homocysteine (AdoHcy) at 2.8 Å resolution and identify a glutamate residue critical for its monomethylation behavior. TbPRMT7 comprises the conserved methyltransferase and β-barrel domains, an N-terminal extension, and a dimerization arm. The active site at the interface of the N-terminal extension, methyltransferase, and β-barrel domains is stabilized by the dimerization arm of the neighboring protomer, providing a structural basis for dimerization as a prerequisite for catalytic activity. Mutagenesis of active-site residues highlights the importance of Glu181, the second of the two invariant glutamate residues of the double E loop that coordinate the target arginine in substrate peptides/proteins and that increase its nucleophilicity. Strikingly, mutation of Glu181 to aspartate converts TbPRMT7 into a type I PRMT, producing asymmetric dimethylarginine (ADMA). Isothermal titration calorimetry (ITC) using a histone H4 peptide showed that the Glu181Asp mutant has markedly increased affinity for monomethylated peptide with respect to the WT, suggesting that the enlarged active site can favorably accommodate monomethylated peptide and provide sufficient space for ADMA formation. In conclusion, these findings yield valuable insights into the product specificity and the catalytic mechanism of protein arginine methyltransferases and have important implications for the rational (re)design of PRMTs.crystal structure | enzyme catalysis | PRMT | histone methylation | epigenetics P osttranslational modifications of proteins can affect their structure, catalytic activity, and molecular interactions (1). Methylation of the guanidino group of arginine residues represents a prominent subset of these reactions (2). Histone arginine methylation is associated with gene silencing and activation (3); the modification of arginine residues in a variety of nonhistone proteins, including splicing and transcription factors, can regulate their activity (4, 5).Most of the enzymes that catalyze arginine methylation are designated protein arginine methyltransferases (PRMTs) and require the cofactor S-adenosyl-L-methionine (AdoMet) as the methyl donor (6). Four types of arginine methylation products havedimethylarginine (SDMA), and δ-N G -monomethylarginine (6, 7). Accordingly, PRMTs can be categorized into four groups: Type I PRMTs catalyze ADMA formation, type II PRMTs catalyze SDMA formation, type III PRMTs catalyze MMA formation, and type IV PRMTs catalyze δ-N G -monomethylarginine formation. Type I, II, and III PRMTs are widely distributed in nature whereas type IV PRMTs seem to be limited to yeasts and plants (8). Interestingly, whereas type I and II enzymes catalyze MMA production in addition ...

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

“…2B). Importantly, although this enzyme contains the ADMA-producing mutation E181D (25), as well as a Q329A mutation in the THW loop, no ADMA formation was detected. SDMA production catalyzed by the E181D/ Q329A mutant was confirmed by TLC analysis where the radioactive product co-migrated with the non-radioactive SDMA standard (Fig.…”

“…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. Notably, the double mutant E172D/E181D was previously tested and found inactive (Table 1) (25).…”

“…Notably, the double mutant E172D/E181D was previously tested and found inactive (Table 1) (25). The additional substitutions in the six new double mutants included M75A, Q329A, Q329N, W330A, F71A, and I173G, each with the E181D mutation, based on their immediate vicinity to the sulfur atom of AdoHcy from which the methyl group of AdoMet is transferred to the arginine residue in protein and peptide substrates (Fig.…”

“…1) (25). The double E loop is a conserved feature of PRMTs that has been shown to directly interact with the methyl-accepting arginine residue (2,11).…”

“…TbPRMT7 had been initially characterized for a possible role in the transcriptional control of gene expression in this organism (26). Here, we have focused on TbPRMT7 because it displays robust type III activity and has been amenable to structural analysis (25)(26)(27). Within this work, we further examine the effects of key active-site residues on the enzymatic activity of TbPRMT7 through mutagenesis and highly sensitive amino acid analysis techniques to demonstrate the importance of the THW loop (MQW for TbPRMT7) ( Fig.…”