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-Structure paper
Title | Combined roles of ATP and small hairpin RNA in the activation of RIG-I revealed by solution-based analysis. |
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Journal, issue, pages | Nucleic Acids Res, Vol. 46, Issue 6, Page 3169-3186, Year 2018 |
Publish date | Apr 6, 2018 |
![]() | Neelam Shah / Simone A Beckham / Jacqueline A Wilce / Matthew C J Wilce / ![]() |
PubMed Abstract | RIG-I (retinoic acid inducible gene-I) is a cytosolic innate immune protein that senses viral dsRNA with a 5'-triphosphate overhang. Upon interaction with dsRNA a de-repression of the RIG-I CARD ...RIG-I (retinoic acid inducible gene-I) is a cytosolic innate immune protein that senses viral dsRNA with a 5'-triphosphate overhang. Upon interaction with dsRNA a de-repression of the RIG-I CARD domains takes place that ultimately leads to the production of type I interferons and pro-inflammatory cytokines. Here we investigate the RIG-I conformational rearrangement upon interaction with an activating 5'-triphosphate-10-base pair dsRNA hairpin loop (10bp) compared with a less active 5'-triphosphate-8-base pair dsRNA hairpin loop (8bp). We use size-exclusion chromatography-coupled small-angle X-ray scattering (SAXS) and limited tryptic digest experiments to show that that upon binding to 10 bp, but not 8 bp, RIG-I becomes extended and shows greater flexibility, reflecting the release of its CARDs. We also examined the effect of different ATP analogues on the conformational changes of RIG-I/dsRNA complexes. Of the analogues tested, the addition of ATP transition state analogue ADP-AlFx further assisted in the complete activation of RIG-I in complex with 10bp and also to some extent RIG-I bound to 8bp. Together these data provide solution-based evidence for the molecular mechanism of innate immune signaling by RIG-I as stimulated by short hairpin RNA and ATP. |
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Methods | SAS (X-ray synchrotron) |
Structure data | ![]() SASDB78: ![]() SASDB88: ![]() SASDB98: ![]() SASDBA8: ![]() SASDBB8: ![]() SASDBD8: ![]() SASDBE8: ![]() SASDBF8: ![]() SASDBG8: ![]() SASDBH8: ![]() SASDBJ8: ![]() SASDBK8: ![]() SASDBL8: ![]() SASDBM8: ![]() SASDBN8: ![]() SASDBP8: ![]() SASDBQ8: Probable ATP-dependent RNA helicase DDX58 (Full-length RIG-I) plus 8mer hairpin dsRNA (SEC-peak2) ![]() SASDBR8: Probable ATP-dependent RNA helicase DDX58 (Full-length RIG-I), 8mer hairpin dsRNA/AMP-PNP (SECpeak2) ![]() SASDBS8: Probable ATP-dependent RNA helicase DDX58 (Full-length RIG-I) 8mer hairpin dsRNA/ADP-AlFx (SECpeak2) ![]() SASDBT8: Probable ATP-dependent RNA helicase DDX58 (Delta-CARDs RIG-I) plus 8mer hairpin dsRNA (SEC-peak2) ![]() SASDBU8: Probable ATP-dependent RNA helicase DDX58 (Delta-CARDs) plus 8mer hairpin dsRNA/AMP-PNP (SEC-peak2) ![]() SASDBV8: |
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