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'寡核苷酸合成酶（OAS）是一个酶家族，通过赋予对病毒感染的抗性，在哺乳动物先天免疫系统中起着重要作用。干扰素刺激细胞后，会产生潜在的OA，然后通过双链RNA激活。活性OA会产生2'-5'连接的寡腺苷，进而激活RNase L（一种降解细胞和病毒RNA的内核酸酶）。 OAS的结构研究非常有价值，因为它们将洞悉OAS 2'2'2'特异性核苷转移酶反应的机制，以及酶的RNA激活。我们正在完成OAS蛋白的第一个晶体结构测定，而OAS蛋白是无结合底物或激活RNA的潜在酶的蛋白质。该结构揭示了与3'特异性聚合酶的结构相似性。该结构的分析为设计诱变实验的基础提供了测试机械假设的基础，并为持续的结构研究选择了RNA构建体。 从OAS活性位点与3'特异性聚合酶的比较之间，我们假设2'和3'核苷基转移酶反应的机制相似，并且2'特异性可能来自底物的不同位置。具体目的1是研究OAS催化机械的成分，该组件负责其核苷转移酶反应的独特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，并追求与ATP底物，2-5A底物/乘积或类似物结合的OA的晶体结构。这些努力构成了一个小型，独立的项目，可以使用适中的资源进行。从对Apo OAS晶体结构的分析中，我们假设OAS活化需要对RNA结合时可能发生的蛋白质的构象变化。具体目的2是研究OAS识别病毒RNA的机制，以及随后通过双链RNA激活OAS酶的机制。将追捕与激活双链RNA或非激活的单链RNA结合的OA复合物的晶体。 OAS-RNA复合物的这些结晶实验是OAS项目开发的可行性研究，这是主要研究者实验室的新方向。