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

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 细胞质中的病毒RNA被先天性免疫受体RIG-I和MDA5识别，这两种受体几乎在所有细胞类型中都存在，对激活干扰素反应至关重要，干扰素反应限制病毒感染并启动适应性免疫系统。RIG-I和MDA5都识别双链RNA，但具有不同的长度和末端结构偏好。这些多结构域蛋白包含一个解旋酶结构域，其ATPase活性是信号所必需的，以及两个与下游信号蛋白相互作用的N-末端卡结构域。未知的大规模构象变化和寡聚化被认为是在激活后发生的。我们建议使用溶液小角X射线散射(SAXS)结合孤立磁区的高分辨率结构来开发全长MDA5的模型。我们在MDA5中准备了一些删除和截断，其SAXS轮廓与原子结构相结合，将允许指定结构域和全长从头计算伪残基模型的刚体精化。随后的研究将检查RNA结合时和解旋酶结构域的ATPase循环中发生的构象变化，并检查MDA5和RIG-I之间的差异，这些差异可能有助于他们的配体偏好。 NSLS X9工作台II以前的工作(2010年6月)产生了关于MDA5的孤立结构域的信息数据，这些数据指导着一些正在进行的实验。我们建议通过收集MDA5的全长和各种结构域缺失的SAXS数据来扩大我们的工作，以确定低分辨率的多域结构。我们计划收集5个结构的数据，这些结构已经纯化到均一性，浓度适合于结晶学筛选(5-15 mg/ml)。尺寸排斥层析、多角度光散射和/或分析性超速离心法表明，所有构建物均呈单分散状态。此外，我们还发现了两种在生化分析和分析超速离心中产生不同行为的RNA配体，这可能与该蛋白家族的信号机制有关。我们将收集这些配体的全长或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。这些数据对于描述我们的生化观察的未来出版物将是有价值的，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测试的假说，将结构和生化行为与细胞内的信号活动联系起来，在允许纳入我们的出版物的时间框架内。我们的研究还将有助于识别柔性区域，以指导晶体筛选的结构设计。

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.