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

0091260格尔森最近，P.I.合作确定了一个高分辨率的晶体结构的原型核酸糖甲基转移酶与其辅因子和RNA底物复合。这种酶，牛痘病毒蛋白VP 39，在5'端帽结构内核糖的2' OH处甲基化信使RNA。结构数据提供了一个不寻常的机会，以确定潜在的核酸核糖甲基化的化学过程。酶的催化中心与结合的底物和辅因子的原子尺度检查表明一种机制，即通过目标核糖羟基的去质子化产生的带电氧攻击甲基供体辅因子（S-腺苷甲硫氨酸）的三价硫，影响辅因子产物（S-腺苷高半胱氨酸）的置换。尽管VP 39残基赖氨酸175的侧链被很好地定位以实现羟基的去质子化，但赖氨酸175的pKa需要降低约三个数量级。相邻残基精氨酸209的侧链可能导致这种抑制发生，天冬氨酸138可能充当质子汇（假设天冬氨酸138侧链的pKa升高）。为了确定靶羟基是否确实在VP 39的情况下去质子化，NMR将用于研究VP 39结合的同位素富集的RNA底物的羟基去质子化。接下来，在（1）用富含同位素的氨乙基化试剂修饰独特的半胱氨酸-175取代突变体和/或（2）产生其中赖氨酸175、精氨酸209或天冬氨酸138各自包含独特的富含13 C/15 N的二肽的一半的蛋白质之后，将检查VP 39侧链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pKa。

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.