Structural Determinants for the Strict Monomethylation Activity by Trypanosoma brucei Protein Arginine Methyltransferase 7

Abstract

Trypanosoma brucei protein arginine methyltransferase 7 (TbPRMT7) exclusively generates monomethylarginine (MMA), which directs biological consequences distinct from that of symmetric dimethylarginine (SDMA) and asymmetric dimethylarginine (ADMA). However, determinants controlling the strict monomethylation activity are unknown. We present the crystal structure of the TbPRMT7 active core in complex with S-adenosyl-L-homocysteine (AdoHcy) and a histone H4 peptide substrate. In the active site, residues E172, E181, and Q329 hydrogen bond the guanidino group of the target arginine and align the terminal guanidino nitrogen in a position suitable for nucleophilic attack on the methyl group of S-adenosyl-L-methionine (AdoMet). Structural comparisons and isothermal titration calorimetry data suggest that the TbPRMT7 active site is narrower than those of protein arginine dimethyltransferases, making it unsuitable to bind MMA in a manner that would support a second turnover, thus abolishing the production of SDMA and ADMA. Our results present the structural interpretations for the monomethylation activity of TbPRMT7.

Substrate Specificity of Human Protein Arginine Methyltransferase 7 (PRMT7)

“…The type III activity of GST-PRMT7 forming only MMA product has been confirmed with both GST-GAR protein derived from fibrillarin and various peptides derived from histone H4, histone H2B, splicing protein SmD3, ribosomal protein RPL3, and HIV-1 TAT protein as well as for PRMT7 automethylation. These results are consistent with previous reports on human and mouse PRMT7 (7,8) as well as studies on the trypanosome PRMT7 (6,9). Although PRMT7 may work synergistically with type I or type II PRMTs or other modulators in vivo, PRMT7 by itself only generates MMA residues.…”

“…Interestingly, trypanosome PRMT7, which contains Gly and Met instead of Asp and Glu at these two positions (Fig. 5), methylates histone H4 at the non-RXR Arg-3 site, indicating that it may have a different substrate specificity from the human or mouse PRMT7 (9). However, it is clear that all of these species are type III PRMTs that only produce MMA as a product.…”

“…Most mammalian PRMTs have been characterized as either type I enzymes that transfer up to two methyl groups from S-adenosyl-L-methionine (AdoMet) to the same terminal nitrogen atom forming -N G -monomethylarginine (MMA) and -N G ,N G -asymmetric dimethylarginine (ADMA) (PRMT1, -2, -3, -4, -6, and -8) or type II enzymes that transfer up to two methyl groups to different terminal nitrogen atoms, forming MMA and -N G ,NЈ Gsymmetric dimethylarginine (SDMA) (PRMT5) (1)(2)(3)(4). However, PRMT7 has been found to produce MMA residues only, which defines it as the first type III PRMT (5)(6)(7)(8)(9). Recent work utilizing an insect cell-expressed mouse PRMT7 demonstrated a unique substrate specificity for RXR motifs among multiple basic residues (8).…”

Transition state mimics are valuable mechanistic probes for structural studies with the arginine methyltransferase CARM1

“…These structures reveal a strong conservation of catalytic domain architecture and specifically the AdoMet binding pocket. In contrast, no structures have been solved for PRMTs in complex with fulllength target proteins, and only a few structures have been reported for PRMTs in complex with small, well-resolved, peptide substrate fragments (18,24,25). The covalent tethering of substrates to enzymes has proven to be an effective strategy for capturing difficult to characterize enzyme-substrate complexes, leading to their structural characterization (26,27).…”

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

“…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.…”