Structure and dynamics of the second CARD of human RIG-I provide mechanistic insights into regulation of RIG-I activation.
RIG-I is a cytosolic sensor of viral RNA, comprised of two N-terminal CARDs followed by helicase and C-terminal regulatory domains (helicase-CTD). Viral RNA binds to the helicase-CTD and "exposes" the CARDs for downstream signaling. The role of the second CARD (CARD2) is essential as RIG-I activation requires dephosphorylation of Thr170 followed by ubiquitination at Lys172. Here, we present the solution structure and dynamics of human RIG-I CARD2. Surprisingly, we find that Thr170 is mostly buried. Parallel studies on the phosphomimetic T170E mutant suggest that the loss of function upon Thr170 phosphorylation is likely associated with changes in the CARD1-CARD2 interface that may prevent Lys172 ubiquitination and/or binding to free K63-linked polyubiquitin. We also demonstrate a strong interaction between CARD2 and the helicase-CTD, and show that mutations at the interface result in constitutive activation of RIG-I. Collectively, our data suggests a close interplay between phosphorylation, ubiquitination, and activation of human RIG-I, all mediated by CARD2.
Ecole Normale Supérieure, Département de Chimie, UMR 7203 CNRS-UPMC-ENS, 24, rue Lhomond, 75005 Paris, France. fabien.ferrage@ens.fr
Fabien
F
Ferrage
Kaushik
K
Dutta
Estanislao
E
Nistal-Villán
Jenish R
JR
Patel
María T
MT
Sánchez-Aparicio
Pablo
P
De Ioannes
Angeliki
A
Buku
Gloria González
GG
Aseguinolaza
Adolfo
A
García-Sastre
Aneel K
AK
Aggarwal
D019943:Amino Acid Substitution; D020134:Catalytic Domain; D000071457:DEAD Box Protein 58; D053487:DEAD-box RNA Helicases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D016899:Interferon-beta / Q000235:genetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D019906:Nuclear Magnetic Resonance, Biomolecular; D010750:Phosphoproteins / Q000737:chemistry; D010766:Phosphorylation; D011401:Promoter Regions, Genetic; D054730:Protein Interaction Domains and Motifs; D017433:Protein Structure, Secondary; D011971:Receptors, Immunologic; D013499:Surface Properties; D015533:Transcriptional Activation; D054875:Ubiquitination
D010750:Phosphoproteins; D011971:Receptors, Immunologic; D016899:Interferon-beta; C452820:RIGI protein, human; D000071457:DEAD Box Protein 58; D053487:DEAD-box RNA Helicases