Structure-function studies of an antiviral enzyme

描述(申请人提供)：2‘-5’寡腺苷合成酶(OAS)是一个酶家族，通过赋予对病毒感染的抵抗力，在哺乳动物的先天免疫系统中发挥重要作用。当干扰素刺激细胞时，潜伏的OAS被产生，随后被双链RNA激活。活跃的OAS产生2‘-5’连接的寡腺苷二聚体并激活核糖核酸酶L，核糖核酸酶是一种降解细胞和病毒核糖核酸的内切核酸酶。OAS的结构研究是有价值的，因为它们将为OAS 2‘特异的核苷酸转移酶反应和该酶的RNA激活提供深入的机制。我们正在完成OAS蛋白质的第一次晶体结构测定，这是一种没有结合底物或激活RNA的潜伏酶。这种结构显示出与3‘-特异性聚合酶的结构相似。对这种结构的分析为设计突变实验以检验机制假说和选择RNA结构进行继续结构研究提供了基础。 通过对OAS活性部位和3‘-特异性聚合酶活性部位的比较，我们推测2’和3‘核苷酸转移酶反应的机制是相似的，2’-特异性可能来自底物的不同位置。具体目的1是研究OAS催化机械的部件，这些部件负责其核苷酸转移酶反应独特的2‘特异性。这将通过观察设计的突变体的功能后果，其中建议的活性部位氨基酸残基已被取代，并通过追求与ATP底物、2-5A底物/产物或类似物结合的OAS的晶体结构来实现。这些努力构成了一个小型、自给自足的项目，可以用不多的资源进行。从对apo OAS晶体结构的分析中，我们假设OAS的激活需要蛋白质的构象变化，这可能发生在RNA结合时。具体目的2是研究OAS识别病毒RNA的机制，以及双链RNA随后激活OAS酶的机制。将寻求与激活的双链RNA或非激活的单链RNA结合的OAS络合物的晶体。这些对OAS-RNA复合体的结晶实验是OAS项目发展的可行性研究，这是首席研究员实验室的一个新方向。

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.