Catalytic Mechanism of Vaccinia Virus Protein in VP39 in 2'-O-Methylation of the mRNA 5' Cap Structure

痘苗病毒蛋白VP39对mRNA 5帽结构2-O-甲基化的催化机制

• 批准号: 0091260
• 负责人: Paul Gershon
• 金额: $ 33万
• 依托单位: Texas A&M Research Foundation
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-04-01 至 2005-03-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0091260&HistoricalAwards=false
• 关键词: Catalytic; Mechanism; Vaccinia; Virus; Protein

0091260GershonRecently, the P.I. collaborated in determining a high-resolution crystal structure for a prototypical nucleic acid sugar methyltransferase complexed with its cofactor and RNA substrate. This enzyme, vaccinia virus protein VP39, methylates messenger RNA at the 2'OH of a ribose within the 5' end cap structure. The structural data afford an unusual opportunity to identify the chemical processes underlying nucleic acid ribose methylation. Atomic-scale examination of the enzyme's catalytic center with bound substrate and cofactor suggests a mechanism whereby the charged oxygen generated by deprotonation of the target ribose hydroxyl attacks the trivalent sulfur of the methyl-donating cofactor (S-adenosylmethionine), effecting displacement of cofactor-product (S-adenosylhomocysteine). Although the sidechain of VP39 residue Lysine 175 is well-positioned to effect deprotonation of the hydroxyl, lysine 175's pKa would need to be depressed ~three orders of magnitude. The sidechain of a neighboring residue, arginine 209 may cause this depression to occur, and aspartate 138 may act as the proton sink (assuming a pKa elevation in the aspartate 138 sidechain). To determine whether the target hydroxyl is indeed deprotonated in the context of VP39, NMR will be used to investigate hydroxyl-deprotonation of the VP39-bound isotope-enriched RNA substrate. Next, VP39 sidechain pKa values will be examined after (1) modification of a unique-cysteine-175 substitution mutant with an isotope-enriched aminoethylation reagent and/or (2) generating proteins in which lysine 175, arginine 209 or aspartate 138 each comprise half of a uniquely 13C/15N enriched dipeptide. In combination with mutagenesis, these selective labeling strategies will facilitate the determination of sidechain pKa in the presence and absence of cofactor-product.

最近，P.I.合作确定了一个典型的核酸糖甲基转移酶的高分辨晶体结构，该酶与其辅因子和RNA底物形成了络合物。这种酶，痘苗病毒蛋白VP39，在5‘端帽结构中核糖的2’OH处甲基化信使RNA。这些结构数据为确定核酸核糖甲基化的化学过程提供了一个不同寻常的机会。结合底物和辅因子的酶催化中心的原子尺度分析表明，目标核糖羟基去质子化产生的电荷氧攻击甲基供体辅因子(S-腺苷蛋氨酸)的三价硫，从而影响辅因子产物(S-腺苷同型半胱氨酸)的置换。虽然VP39残基赖氨酸175的侧链很好地实现了羟基的去质子化，但赖氨酸175‘S的pKA需要被抑制三个数量级。邻近残基精氨酸209的侧链可能导致这种抑制的发生，并且天冬氨酸138可以充当质子汇(假设天冬氨酸138侧链中的pKA升高)。为了确定目标羟基在VP39的背景下是否确实被去质子化，将使用核磁共振来研究VP39结合的同位素丰富的RNA底物的羟基去质子化。接下来，将在(1)用同位素富集胺乙基化试剂修饰唯一的半胱氨酸-175替代突变体和/或(2)产生赖氨酸175、精氨酸209或天冬氨酸138各自包含独特的13C/15N富集二肽的一半的蛋白质后，检测VP39侧链蛋白的pKa值。结合诱变，这些选择性标记策略将有助于在存在和不存在辅因子产物的情况下测定侧链蛋白PKA。