Abstract
RIG-I is a cytosolic helicase that senses 5′-ppp RNA contained in negative-strand RNA viruses and triggers innate antiviral immune responses. Calorimetric binding studies established that the RIG-I C-terminal regulatory domain (CTD) binds to blunt-end double-stranded 5′-ppp RNA a factor of 17 more tightly than to its single-stranded counterpart. Here we report on the crystal structure of RIG-I CTD bound to both blunt ends of a self-complementary 5′-ppp dsRNA 12-mer, with interactions involving 5′-pp clearly visible in the complex. The structure, supported by mutation studies, defines how a lysine-rich basic cleft within the RIG-I CTD sequesters the observable 5′-pp of the bound RNA, with a stacked phenylalanine capping the terminal base pair. Key intermolecular interactions observed in the crystalline state are retained in the complex of 5′-ppp dsRNA 24-mer and full-length RIG-I under in vivo conditions, as evaluated from the impact of binding pocket RIG-I mutations and 2′-OCH3 RNA modifications on the interferon response.
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Acknowledgements
The authors are grateful to the staff of X-29 beamline at Brookhaven National Laboratory for their assistance during synchrotron data collection, D. Shimanshu and Y. Tian for their assistance in synchrotron data collection, M. Aigner for chemical synthesis of 5′-ppp RNAs undertaken in Innsbruck and G. Wardle for MS analysis in the RU proteomics facility to quality control the 5′-ppp RNAs. D.J.P. was supported by funds from the Abby Rockefeller Mauze Trust and the Maloris Foundation. R.M. was supported by the Austrian Science fund FWF (I317). G.H. was supported by grants from the Bundesministerium für Bildung und Forschung Biofuture and GoBio and from the Deutsche Forschungsgemeinschaft (SFB704, SFB670, SFB832 and KFO177).
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Y.W. expressed the RIG-I CTD domain, with G.S. assisting with protein expression and purification; Y.W. crystallized and solved the structure of the complex; J.L. was responsible for chemical synthesis of palindromic and non-palindromic 5′-ppp RNAs and 2′-O-methyl-modified 5′-ppp RNAs; C.S., M.G. and M.S. were responsible for the in vivo functional assays, including expression of full-length RIG-I mutants; H.L. and Y.W. were responsible for ITC titration assays; S.J. was responsible for gel-shift assays and prepared the plasmids for mutant RIG-I CTD expression; R.M. provided earlier batches of short 5′-ppp RNAs for crystallization trials; the paper was written by D.J.P., G.H. and T.T. with the assistance of the other authors.
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Wang, Y., Ludwig, J., Schuberth, C. et al. Structural and functional insights into 5′-ppp RNA pattern recognition by the innate immune receptor RIG-I. Nat Struct Mol Biol 17, 781–787 (2010). https://doi.org/10.1038/nsmb.1863
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DOI: https://doi.org/10.1038/nsmb.1863
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