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TitleCombined roles of ATP and small hairpin RNA in the activation of RIG-I revealed by solution-based analysis.
Journal, issue, pagesNucleic Acids Res, Vol. 46, Issue 6, Page 3169-3186, Year 2018
Publish dateApr 6, 2018
AuthorsNeelam Shah / Simone A Beckham / Jacqueline A Wilce / Matthew C J Wilce /
PubMed AbstractRIG-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.
External linksNucleic Acids Res / PubMed:29346611 / PubMed Central
MethodsSAS (X-ray synchrotron)
Structure data

SASDB78:
Probable ATP-dependent RNA helicase DDX58 (Full-length RIG-I)
Method: SAXS/SANS

SASDB88:
Probable ATP-dependent RNA helicase DDX58 (Full-length RIG-I) plus ADP-AlFx
Method: SAXS/SANS

SASDB98:
Probable ATP-dependent RNA helicase DDX58 (Full-length RIG-I) plus bound 10mer hairpin dsRNA
Method: SAXS/SANS

SASDBA8:
Probable ATP-dependent RNA helicase DDX58 (Full-length RIG-I) plus 10mer hairpin dsRNA /AMP-PNP
Method: SAXS/SANS

SASDBB8:
Probable ATP-dependent RNA helicase DDX58 (Full-length RIG-I) plus 10mer hairpin dsRNA and ADP-AlFx
Method: SAXS/SANS

SASDBD8:
Probable ATP-dependent RNA helicase DDX58 (Full-length RIG-I) plus 8mer hairpin dsRNA (SEC-peak1)
Method: SAXS/SANS

SASDBE8:
Probable ATP-dependent RNA helicase DDX58 (Full-length RIG-I) 8mer hairpin dsRNA/AMP-PNP (SEC-peak1)
Method: SAXS/SANS

SASDBF8:
Probable ATP-dependent RNA helicase DDX58 (Full-length RIG-I) 8mer hairpin dsRNA/ADP-AlFx (SECpeak1)
Method: SAXS/SANS

SASDBG8:
Probable ATP-dependent RNA helicase DDX58 without CARDs (Delta-CARDs RIG-I)
Method: SAXS/SANS

SASDBH8:
Probable ATP-dependent RNA helicase DDX58 without CARDs (Delta-CARDs RIG-I) plus ADP-AlFx
Method: SAXS/SANS

SASDBJ8:
Probable ATP-dependent RNA helicase DDX58 without CARDs (Delta-CARDs RIG-I) plus 10mer hairpin dsRNA
Method: SAXS/SANS

SASDBK8:
Probable ATP-dependent RNA helicase DDX58 (Delta-CARDs RIG-I) plus 10mer hairpin dsRNA and AMP-PNP
Method: SAXS/SANS

SASDBL8:
Probable ATP-dependent RNA helicase DDX58 (Delta-CARDs RIG-I) plus 10mer hairpin dsRNA and ADP-AlFx
Method: SAXS/SANS

SASDBM8:
Probable ATP-dependent RNA helicase DDX58 (Delta-CARDs RIG-I) plus 8mer hairpin dsRNA (SEC-peak1)
Method: SAXS/SANS

SASDBN8:
Probable ATP-dependent RNA helicase DDX58 (Delta-CARDs) plus 8mer hairpin dsRNA/AMP-PNP (SEC-peak1)
Method: SAXS/SANS

SASDBP8:
Probable ATP-dependent RNA helicase DDX58 (Delta-CARDs) plus 8mer hairpin dsRNA/ADP-AlFx (SEC-peak1)
Method: SAXS/SANS

SASDBQ8: Probable ATP-dependent RNA helicase DDX58 (Full-length RIG-I) plus 8mer hairpin dsRNA (SEC-peak2)
Method: SAXS/SANS

SASDBR8: Probable ATP-dependent RNA helicase DDX58 (Full-length RIG-I), 8mer hairpin dsRNA/AMP-PNP (SECpeak2)
Method: SAXS/SANS

SASDBS8: Probable ATP-dependent RNA helicase DDX58 (Full-length RIG-I) 8mer hairpin dsRNA/ADP-AlFx (SECpeak2)
Method: SAXS/SANS

SASDBT8: Probable ATP-dependent RNA helicase DDX58 (Delta-CARDs RIG-I) plus 8mer hairpin dsRNA (SEC-peak2)
Method: SAXS/SANS

SASDBU8: Probable ATP-dependent RNA helicase DDX58 (Delta-CARDs) plus 8mer hairpin dsRNA/AMP-PNP (SEC-peak2)
Method: SAXS/SANS

SASDBV8:
Probable ATP-dependent RNA helicase DDX58 (Delta-CARDs) plus 8mer hairpin dsRNA/ADP-AlFx (SEC-peak2)
Method: SAXS/SANS

Source
  • Homo sapiens (human)

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