The Structural And Enzymatic Investigations Of Protein Arginine Methyltransferase6and7from Trypanosoma Brucei

Arginine methylation is a widespread posttranslational modification that plays important roles in transcription regulation, RNA processing, DNA repair, and signal transduction. Protein arginine methyltransferases (PRMT) are a family of enzymes that catalyze the methylation on the guanidino nitrogen atoms of protein arginyl residues, utilizing S-adenosyl-L-methionine (AdoMet) as a methyl donor. Based on the kind of methylated arginine products formed, PRMTs are classified into three types.Arginine methylation plays vital roles in the cellular functions of the protozoan Trypanosoma brucei. The T. brucei arginine methyltransferase6(TbPRMT6) is a type I arginine methyltransferase homologous to human PRMT6. In this study, we report the crystal structures of apo-TbPRMT6and its complex with the reaction product S-adenosyl-homocysteine (SAH). The structure of apo-TbPRMT6displays several features that are different from those of type I PRMTs that were structurally characterized previously, including four stretches of insertion, the absence of strand β15, and a distinct dimerization arm. The comparison of the apo-TbPRMT6and SAH-TbPRMT6structures revealed the fine rearrangements in the active site upon SAH binding. The isothermal titration calorimetry results demonstrated that SAH binding greatly increases the affinity of TbPRMT6to a substrate peptide derived from bovine histone H4. The western blotting and mass spectrometry results revealed that TbPRMT6methylates bovine histone H4tail at arginine3but cannot methylate several T. brucei histone tails. In summary, our results highlight the structural differences between TbPRMT6and other type I PRMTs and reveal that the active site rearrangement upon SAH binding is important for the substrate binding of TbPRMT6.TbPRMT7, which is suggested to be the T. brucei homolog of HsPRMT7, is the first clearly characterized type III PRMT. Most importantly, TbPRMT7lacks the second AdoMet-binding-like domain and shows robust in vitro activity, providing a tractable system to study the unique product specificity of the class III arginine methyltransferases. To understand the origins for the strict monomethylation activity of TbPRMT7, we determined the structure of the TbPRMT7enzymatic core in complex with AdoHcy and H41-21. In the active site, residues E172, E181and Q329form hydrogen bonds with 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 AdoMet. Structural comparisons and isothermal titration calorimetry data suggest that the TbPRMT7active 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.