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.
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