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.

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 细胞质中的病毒RNA被先天免疫受体RIG-I和MDA 5识别，这些受体在几乎所有细胞类型中发现，并且对于激活限制病毒感染和引发适应性免疫系统的干扰素应答至关重要。Rig-I和MDA 5都识别双链RNA，但具有不同的长度和末端结构偏好。这些多结构域蛋白含有解旋酶结构域，其ATP酶活性是信号传导所需的，以及两个N-末端CARD结构域，其与下游信号传导蛋白相互作用。已经提出在活化时发生未知的大规模构象变化和寡聚化。我们建议使用解决方案小角X射线散射（SAXS）结合高分辨率结构的孤立域开发一个模型的全长MDA 5。我们已经准备了一些删除和截断MDA 5的SAXS配置文件结合原子结构将允许分配域和刚体细化的全长从头算虚拟残基模型。随后的研究将检查RNA结合时和解旋酶结构域的ATP酶循环期间发生的构象变化，并检查可能导致其配体偏好的MDA 5和Rig-I之间的差异。 在NSLS X9工作台II（2010年6月）的先前工作导致了MDA 5的孤立结构域的信息数据，这些结构未知，指导了许多正在进行的实验。我们建议扩大我们的工作，收集SAXS数据的全长和各种域缺失的MDA 5，以确定一个低分辨率的多域结构。我们计划收集关于5种构建体的数据，这些构建体已经纯化至均一性和适合于晶体学筛选的浓度（5- 15 mg/ml）。如尺寸排阻色谱、多角度光散射和/或分析超速离心所示，所有构建体均呈单分散性。此外，我们还发现了两种RNA配体，它们在生化测定和分析超电泳中产生不同的行为，这可能与该蛋白质家族的信号传导机制有关。我们将收集具有这些配体的全长或CARD缺失蛋白的SAXS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。这些数据将是有价值的未来出版物描述我们的生化观察，我们的目标是在2011年下半年提交。 快速访问将帮助我们设计可验证的假设，将结构和生化行为与细胞内的信号传导活动联系起来，并在允许包含在我们的出版物中的时间范围内。 我们的研究也将有助于确定灵活的区域，以指导晶体学筛选的结构设计。