Structure-function studies of an antiviral enzyme

抗病毒酶的结构功能研究

• 批准号: 6726795
• 负责人: VIVIEN YEE
• 金额: $ 7.65万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-05-01 至 2006-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6726795
• 关键词: RNA binding protein; adenosine monophosphate; catalyst; crystallization; enzyme activity; enzyme induction /repression; enzyme substrate; immunity; intermolecular interaction; nucleic acid structure; oligonucleotides; protein structure function; virus diseases

DESCRIPTION (provided by applicant): The 2'-5' oligoadenylate synthetases (OAS) are a family of enzymes which play an important role in the mammalian innate immune system by conferring resistance to viral infections. Upon interferon stimulation of cells, latent OAS is produced and subsequently activated by double-stranded RNA. Active OAS produces 2'-5' linked oligoadenosines which in turn dimerize and activate RNase L, an endoribonuclease that degrades cellular and viral RNA. Structural studies of OAS are valuable since they will provide insight into the mechanisms for the OAS 2'-specific nucleotidyl transferase reaction, and for the RNA activation of the enzyme. We are in the process of completing the first crystal structure determination of an OAS protein, that of a latent enzyme without bound substrate or activating RNA. This structure reveals a structural similarity with 3'-specific polymerases. Analysis of this structure provides a basis for designing mutagenesis experiments to test mechanistic hypotheses, and for selecting RNA constructs for continuing structural studies. From the comparison between the OAS active site and those in 3'-specific polymerases, we hypothesize that the mechanisms for the 2' and 3' nucleotidyl transferase reactions are similar, and that the 2' specificity may arise from a differing position of the substrate. Specific Aim 1 is to investigate the components of the OAS catalytic machinery which are responsible for the unique 2' specificity of its nucleotidyl transferase reaction. This will be done by observing the functional consequences of designed mutants in which proposed active site amino acid residues have been substituted, and by pursuing crystal structures of OAS bound to ATP substrate, 2-5A substrate/product, or analogs. These efforts constitute a small, self-contained project which can be carried out with modest resources. From the analysis of our apo OAS crystal structure, we hypothesize that OAS activation requires a conformational change of the protein which likely occurs upon RNA binding. Specific Aim 2 is to investigate the mechanism of OAS recognition of viral RNA, and of the subsequent activation of the OAS enzyme by double-stranded RNA. Crystals of complexes of OAS bound to activating double-stranded RNA, or to non-activating single-stranded RNA, will be pursued. These crystallization experiments for OAS-RNA complexes are feasibility studies for the development of the OAS project, which is a new direction for the Principal Investigator's laboratory.

描述（由申请人提供）：2'-5'寡腺苷酸合成酶（OAS）是一个酶家族，其通过赋予对病毒感染的抵抗力而在哺乳动物先天免疫系统中发挥重要作用。细胞受到干扰素刺激后，会产生潜在的 OAS，随后被双链 RNA 激活。活性 O​​AS 产生 2'-5' 连接的寡腺苷，进而二聚化并激活 RNase L（一种降解细胞和病毒 RNA 的核糖核酸内切酶）。 OAS 的结构研究很有价值，因为它们将深入了解 OAS 2' 特异性核苷酸转移酶反应的机制以及该酶的 RNA 激活机制。我们正在完成 OAS 蛋白的首次晶体结构测定，该蛋白是一种没有结合底物或激活 RNA 的潜在酶。该结构揭示了与 3' 特异性聚合酶的结构相似性。对该结构的分析为设计诱变实验以测试机制假设以及为继续结构研究选择 RNA 构建体提供了基础。 通过比较 OAS 活性位点和 3' 特异性聚合酶中的活性位点，我们假设 2' 和 3' 核苷酸基转移酶反应的机制相似，并且 2' 特异性可能源于底物的不同位置。具体目标 1 是研究 OAS 催化机制的组成部分，这些组成部分负责其核苷酸基转移酶反应的独特 2' 特异性。这将通过观察设计的突变体的功能后果来完成，其中所提出的活性位点氨基酸残基已被取代，并通过研究与 ATP 底物、2-5A 底物/产物或类似物结合的 OAS 的晶体结构。这些努力构成了一个小型、独立的项目，只需少量资源即可完成。通过对 apo OAS 晶体结构的分析，我们假设 OAS 激活需要蛋白质的构象变化，这可能在 RNA 结合时发生。具体目标 2 是研究 OAS 识别病毒 RNA 以及随后双链 RNA 激活 OAS 酶的机制。将寻找与活化双链RNA或非活化单链RNA结合的OAS复合物晶体。这些OAS-RNA复合物的结晶实验是OAS项目发展的可行性研究，也是首席研究员实验室的一个新方向。