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. º The structure of a CARD of human RIG-I º The mechanism and structural role of phosphorylation are revealed º In trans interaction with the Helicase and regulatory domains is shown º Lys172 lies in proximity to the CARD2:helicase-CTD interface RIG-I is a cytosolic receptor with a role in mediating innate immunity, with two N-terminal CARD domains that get exposed upon viral RNA binding. Ferrage et al. study the structure and dynamics of the second CARD and examine roles of phosphorylation in the regulation of RIG-I activation in response to viral infection.