Agonist and antagonist recognition by RIG-I, a cytoplasmic innate immunity receptor.
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.
Ranjith-Kumar
C T
CT
Department of Biochemistry and Biophysics, Texas A&M University, College Station, Texas 77843-2128, USA. ctrkumar@gmail.com
Murali
Ayaluru
A
Dong
Wen
W
Srisathiyanarayanan
Dharmaiah
D
Vaughan
Robert
R
Ortiz-Alacantara
Joanna
J
Bhardwaj
Kanchan
K
Li
Xiaojun
X
Li
Pingwei
P
Kao
Cheng C
CC
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D003593:Cytoplasm / Q000187:drug effects / Q000276:immunology* / Q000378:metabolism*; D053487:DEAD-box RNA Helicases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D006801:Humans; D007113:Immunity, Innate / Q000187:drug effects / Q000276:immunology*; D008024:Ligands; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011971:Receptors, Immunologic / Q000819:agonists / Q000037:antagonists & inhibitors / Q000276:immunology*; D015398:Signal Transduction / Q000187:drug effects; D018411:Spodoptera
D008024:Ligands; D011971:Receptors, Immunologic; D053487:DEAD-box RNA Helicases