Dolzhanskaya Natalia, Bolton David C, Denman Robert B
Department of Molecular Biology, New York State Institute for Basic Research in Developmental Disabilities, 1050 Forest Hill Road, Staten Island, New York 10314, USA.
Biochemistry. 2008 Aug 19;47(33):8491-503. doi: 10.1021/bi702298f. Epub 2008 Jul 26.
Exon 15 of the fragile X mental retardation protein gene (FMR1) is alternatively spliced into three variants. The amino acids encoded by the 5' end of the exon contain several regulatory determinants including phosphorylation sites and a potential conformational switch. Residues encoded by the 3' end of the exon specify FMRP's RGG box, an RNA binding domain that interacts with G-quartet motifs. Previous studies demonstrated that the exon 15-encoded N-terminal residues influence the extent of arginine methylation, independent of S 500 phosphorylation. In the present study we focus on the role the putative conformational switch plays in arginine methylation. Chemical and structural probing of Ex15 alternatively spliced variant proteins and several mutants leads to the following conclusions: Ex15c resides largely in a conformation that is refractory toward methylation; however, it can be methylated by supplementing extracts with recombinant PRMT1 or PRMT3. Protein modeling studies reveal that the RG-rich region is part of a three to four strand antiparallel beta-sheet, which in other RNA binding proteins functions as a platform for nucleic acid interactions. In the Ex15c variant the first strand of this sheet is truncated, and this significantly perturbs the side-chain conformations of the arginine residues in the RG-rich region. Mutating R 507 in the conformational switch to K also truncates the first strand of the beta-sheet, and corresponding decreases in in vitro methylation were found for this and R 507/R 544 and R 507/R 546 double mutants. These effects are not due to the loss of R 507 methylation as a conformational switch-containing peptide reacted under substrate excess and in methyl donor excess was not significantly methylated. Consistent with this, similar changes in beta-sheet structure and decreases in in vitro methylation were observed with a W 513-K mutant. These data support a novel model for FMRP arginine methylation and a role for conformational switch residues in arginine modification.
脆性X智力低下蛋白基因（FMR1）的外显子15可选择性剪接为三种变体。该外显子5'端编码的氨基酸包含几个调控决定因素，包括磷酸化位点和一个潜在的构象开关。外显子3'端编码的残基确定了FMRP的RGG框，这是一个与G-四重体基序相互作用的RNA结合结构域。先前的研究表明，外显子15编码的N端残基影响精氨酸甲基化的程度，与S500磷酸化无关。在本研究中，我们关注假定的构象开关在精氨酸甲基化中所起的作用。对Ex15选择性剪接变体蛋白和几个突变体进行化学和结构探测，得出以下结论：Ex15c主要以一种对甲基化具有抗性的构象存在；然而，通过用重组PRMT1或PRMT3补充提取物，它可以被甲基化。蛋白质建模研究表明，富含RG的区域是三到四条链反平行β-折叠的一部分，在其他RNA结合蛋白中，它作为核酸相互作用的平台。在Ex15c变体中，这片层的第一条链被截断，这显著扰乱了富含RG区域中精氨酸残基的侧链构象。将构象开关中的R507突变为K也会截断β-折叠的第一条链，并且发现该突变体以及R507/R544和R507/R546双突变体的体外甲基化相应降低。这些效应不是由于R507甲基化的丧失，因为在底物过量和甲基供体过量的情况下反应的含构象开关的肽没有被显著甲基化。与此一致，用W513-K突变体观察到β-折叠结构的类似变化和体外甲基化的降低。这些数据支持了一个关于FMRP精氨酸甲基化的新模型以及构象开关残基在精氨酸修饰中的作用。
