A glutamate/aspartate switch controls product specificity in a protein arginine methyltransferase
A glutamate/aspartate switch controls product specificity in a protein arginine methyltransferase
复制标题
DOI：
10.1073/pnas.1525783113
复制
发表时间：
2016-02-23
影响因子：
11.1
通讯作者：
Stavropoulos, Pete
中科院分区：
文献类型：
--
作者：
Debler, Erik W.;Jain, Kanishk;Stavropoulos, Pete
布氏锥虫PRMT 7（TbPRMT 7）是一种蛋白质精氨酸甲基转移酶（PRMT），其严格地使各种底物单甲基化，因此将其分类为III型PRMT。然而，其独特的产品特异性的分子基础仍然难以捉摸。在这里，我们提出了TbPRMT 7的结构与其辅因子产物S-腺苷-L-同型半胱氨酸（HCY）在2.8埃分辨率的复合物，并确定其单甲基化行为的关键谷氨酸残基。TbPRMT 7包含保守的甲基转移酶和β-桶结构域、N-末端延伸和二聚化臂。在N-末端延伸、甲基转移酶和β-桶结构域的界面处的活性位点由相邻原聚体的二聚化臂稳定，为二聚化提供结构基础作为催化活性的先决条件。活性位点残基的诱变突出了Glu 181的重要性，Glu 181是双E环的两个不变的谷氨酸残基中的第二个，其协调底物肽/蛋白质中的靶精氨酸并增加其亲核性。引人注目的是，Glu 181突变为天冬氨酸将TbPRMT 7转化为I型PRMT，产生不对称二甲基精氨酸（ADMA）。使用组蛋白H4肽的等温滴定量热法（ITC）显示，Glu 181 Asp突变体相对于WT对单甲基化肽具有显著增加的亲和力，这表明扩大的活性位点可以有利地容纳单甲基化肽并为ADMA形成提供足够的空间。总之，这些研究结果产生有价值的见解的产品特异性和蛋白质精氨酸甲基转移酶的催化机制，并有重要的影响，PRMT的合理（重新）设计。
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 angstrom resolution and identify a glutamate residue critical for its monomethylation behavior. TbPRMT7 comprises the conserved methyltransferase and beta-barrel domains, an N-terminal extension, and a dimerization arm. The active site at the interface of the N-terminal extension, methyltransferase, and beta-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.