SOLUTION STRUCTURE STUDIES OF MDA5 AND OTHER RIG-I LIKE RECEPTORS

MDA5 和其他 RIG-I 样受体的溶液结构研究

• 批准号: 8363548
• 负责人: Yorgo Modis
• 金额: $ 45.49万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-07-01 至 2012-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8363548
• 关键词: ATP phosphohydrolase; Behavior; Biochemical; Biological Assay; Cells; Cytoplasm; Data; Double-Stranded RNA; Funding; Future; Grant; Immune system; Immunologic Receptors; Interferons; Length; Ligands; Link; Modeling; Molecular Sieve Chromatography; N-terminal; National Center for Research Resources; Principal Investigator; Protein Family; Proteins; Publications; RNA; RNA Binding; Research; Research Infrastructure; Resolution; Resources; Roentgen Rays; Screening procedure; Signal Transduction; Signaling Protein; Solutions; Source; Structure; Time; United States National Institutes of Health; Virus Diseases; Work; analytical ultracentrifugation; cell type; collected works; cost; design; design and construction; flexibility; helicase; light scattering; preference; receptor; research study; response; viral RNA

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Viral RNA in the cytoplasm is recognized by the innate immune receptors RIG-I and MDA5 which are found in nearly all cell types and are critical for activating interferon responses that limit viral infection and prime the adaptive immune system. Both Rig-I and MDA5 recognize double stranded RNA but with different length and end structure preferences. These multidomain proteins contain a helicase domain whose ATPase activity is required for signaling as well as two N-terminal CARD domains that interact with downstream signaling proteins. Unknown large scale conformational changes and oligomerization have been proposed to occur upon activation. We propose to use solution Small Angle X-ray Scattering (SAXS) combined with high resolution structures of isolated domains to develop a model for full-length MDA5. We have prepared a number of deletions and truncations in MDA5 whose SAXS profiles in conjunction with atomic structures will allow the assignment of domains and rigid body refinement of a full-length ab initio dummy-residue model. Subsequent studies will examine the conformational changes that occur upon RNA binding and during the ATPase cycle of the helicase domain, and examine differences between MDA5 and Rig-I that may contribute to their ligand preferences. Previous work at the NSLS X9 workbench II (June 2010) resulted in informative data for isolated domains of MDA5 with no known structure that are guiding a number of ongoing experiments. We propose to expand our work by collecting SAXS data for full length and various domain deletions of MDA5 to determine a low resolution multidomain structure. We plan to collect data on 5 constructs which have been purified to homogeneity and concentrations suitable for crystallographic screening (5-15mg/ml). All constructs appear monodisperse as shown by size exclusion chromatography, multi-angle light scattering and/or analytical ultracentrifugation. In addition, we have discovered two RNA ligands that generate different behaviors in biochemical assays and analytical ultracentrifugation which may be related to this protein family's signaling mechanism. We will collect SAXS data of full-length or CARD deleted protein with these ligands to determine if their biochemical behavior can be explained by different conformational changes. This data will be valuable for a future publication describing our biochemical observations which we aim to submit in the second half of 2011. Rapid access will assist us in designing testable hypotheses that link structure and biochemical behavior to signaling activity within cells in a time frame that allows for inclusion in our publication. Our studies will also facilitate the identification of flexible regions to guide construct design for crystallographic screening.

该副本是利用资源的众多研究子项目之一 由NIH/NCRR资助的中心赠款提供。对该子弹的主要支持 而且，副投影的主要研究员可能是其他来源提供的 包括其他NIH来源。 列出的总费用可能 代表subproject使用的中心基础架构的估计量， NCRR赠款不直接向子弹或副本人员提供的直接资金。 细胞质中的病毒RNA被先天免疫受体IG-I和MDA5识别，它们几乎在所有细胞类型中都发现，对于激活限制病毒感染并启动适应性免疫系统的干扰素反应至关重要。 RIG-I和MDA5都识别双链RNA，但具有不同的长度和终端结构偏好。这些多域蛋白包含一个解旋酶结构域，其ATPase活性是信号传导以及两个与下游信号蛋白相互作用的N末端卡域。激活后已经提出了未知的大规模构象变化和寡聚化。我们建议使用溶液小角度X射线散射（SAXS）与孤立域的高分辨率结构相结合，以开发全长MDA5的模型。我们已经在MDA5中准备了许多删除和截断，其萨克斯剖面与原子结构结合使用，将允许分配域和刚体的刚性体现。随后的研究将检查RNA结合和解旋酶结构域的ATPase周期上发生的构象变化，并检查MDA5和RIG-I之间可能有助于其配体偏好的差异。 NSLS X9 Workbench II（2010年6月）的先前工作为MDA5的隔离域提供了信息，没有已知的结构指导许多正在进行的实验。我们建议通过收集全长的SAXS数据和MDA5的各种域删除来扩展我们的工作，以确定低分辨率的多域结构。我们计划收集有关已纯化为均匀性和适合晶体学筛选（5-15mg/mL）的浓度的5种结构的数据。所有构建体均显示单分散，如大小排除色谱，多角度光散射和/或分析性超速离心。此外，我们发现了两个RNA配体在生化测定和分析性超速离心中产生不同的行为，这可能与该蛋白质家族的信号传导机制有关。我们将使用这些配体收集全长或卡删除蛋白的SAX数据，以确定它们的生化行为是否可以通过不同的构象变化来解释。这些数据对于未来的出版物来说将是有价值的，该出版物描述了我们旨在在2011年下半年提交的生化观察结果。快速访问将有助于我们设计可测试的假设，这些假设将结构和生化行为与电池中的信号活动联系起来，以便在我们的出版物中纳入细胞中的信号活动。 我们的研究还将促进对柔性区域的识别，以指导构造设计进行晶体学筛查。