Structure-function studies of an antiviral enzyme

描述（由申请人提供）：2 '-5'寡腺苷酸合成酶（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发挥重要作用。在干扰素刺激细胞后，产生潜伏的OAS，随后被双链RNA激活。活性OAS产生2 '-5'连接的寡腺苷，其继而二聚化并激活RNase L，RNase L是一种降解细胞和病毒RNA的核糖核酸内切酶。OAS的结构研究是有价值的，因为它们将为OAS 2 '特异性核苷酸转移酶反应和该酶的RNA活化机制提供深入了解。我们正在完成一种OAS蛋白的第一个晶体结构测定，这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。这种结构的分析提供了一个基础，设计诱变实验，以测试机制的假设，并选择RNA构建体继续结构研究。 从OAS活性位点和3 '-特异性聚合酶活性位点之间的比较，我们假设2'和3'核苷酸转移酶反应的机制是相似的，并且2'特异性可能来自底物的不同位置。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结果来完成，其中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的晶体结构来完成。这些努力是一个小型、自足的项目，可以用不多的资源来执行。从我们的载脂蛋白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，这可能发生在RNA结合。具体目标2是研究OAS识别病毒RNA的机制，以及OAS酶随后被双链RNA激活的机制。将追求与活化双链RNA或非活化单链RNA结合的OAS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，这是首席研究员实验室的新方向。

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.