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Agonist and antagonist recognition by RIG-I, a cytoplasmic innate immunity receptor.

ABSTRACT: Cytoplasmic RNA receptors are important in the detection of and response to viral infections. We analyzed ligand recognition by the retinoic acid-inducible protein I (RIG-I) protein in biochemical assays and in transiently transfected cells and characterized the requirements for both single- and double-stranded RNA agonists for RIG-I activation of signaling. RIG-I mutants such as K270A and T409A/S411A that were defective in signaling with triphosphorylated single-stranded RNAs were perfectly capable of signaling with dsRNAs. Furthermore, phosphorothioated oligodeoxynucleotides were found to antagonize RIG-I signaling. Both agonists and antagonist bind purified RIG-I protein and a truncated RIG-I protein that lacked the signaling domain. The agonists were necessary to activate RIG-I ATPase activity in vitro, whereas antagonist inhibited ATPase activity. Differential scanning fluorometry showed that RIG-I bound to agonists, and antagonists have different denaturation properties, suggesting a difference in protein conformations. Last, single particle reconstruction was used to generate three-dimensional models of the RIG-I dimers in complex with an agonist and an antagonist. The two complexes exhibited dramatically different structures.

SUBMITTER: Ranjith-Kumar CT

PROVIDER: S-EPMC2613625 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Retinoic-acid-inducible gene-I (RIG-I; also known as DDX58) is a cytoplasmic pathogen recognition receptor that recognizes pathogen-associated molecular pattern (PAMP) motifs to differentiate between viral and cellular RNAs. RIG-I is activated by blunt-ended double-stranded (ds)RNA with or without a 5'-triphosphate (ppp), by single-stranded RNA marked by a 5'-ppp and by polyuridine sequences. Upon binding to such PAMP motifs, RIG-I initiates a signalling cascade that induces innate immune defences and inflammatory cytokines to establish an antiviral state. The RIG-I pathway is highly regulated and aberrant signalling leads to apoptosis, altered cell differentiation, inflammation, autoimmune diseases and cancer. The helicase and repressor domains (RD) of RIG-I recognize dsRNA and 5'-ppp RNA to activate the two amino-terminal caspase recruitment domains (CARDs) for signalling. Here, to understand the synergy between the helicase and the RD for RNA binding, and the contribution of ATP hydrolysis to RIG-I activation, we determined the structure of human RIG-I helicase-RD in complex with dsRNA and an ATP analogue. The helicase-RD organizes into a ring around dsRNA, capping one end, while contacting both strands using previously uncharacterized motifs to recognize dsRNA. Small-angle X-ray scattering, limited proteolysis and differential scanning fluorimetry indicate that RIG-I is in an extended and flexible conformation that compacts upon binding RNA. These results provide a detailed view of the role of helicase in dsRNA recognition, the synergy between the RD and the helicase for RNA binding and the organization of full-length RIG-I bound to dsRNA, and provide evidence of a conformational change upon RNA binding. The RIG-I helicase-RD structure is consistent with dsRNA translocation without unwinding and cooperative binding to RNA. The structure yields unprecedented insight into innate immunity and has a broader impact on other areas of biology, including RNA interference and DNA repair, which utilize homologous helicase domains within DICER and FANCM.

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Agonist and antagonist recognition by RIG-I, a cytoplasmic innate immunity receptor.

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