Dataset Information

Structure and dynamics of the second CARD of human RIG-I provide mechanistic insights into regulation of RIG-I activation.

ABSTRACT: 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.

SUBMITTER: Ferrage F

PROVIDER: S-EPMC3625992 | biostudies-literature | 2012 Dec

REPOSITORIES: biostudies-literature

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Structure and dynamics of the second CARD of human RIG-I provide mechanistic insights into regulation of RIG-I activation.

Ferrage Fabien F Dutta Kaushik K Nistal-Villán Estanislao E Patel Jenish R JR Sánchez-Aparicio María T MT De Ioannes Pablo P Buku Angeliki A Aseguinolaza Gloria González GG García-Sastre Adolfo A Aggarwal Aneel K AK

Structure (London, England : 1993) 20121011 12

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 buri ...[more]

PMID: 23063562

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Project description:There is growing interest in models of regulatory sequence evolution. However, existing models specifically designed for regulatory sequences consider the independent evolution of individual transcription factor (TF)-binding sites, ignoring that the function and evolution of a binding site depends on its context, typically the cis-regulatory module (CRM) in which the site is located. Moreover, existing models do not account for the gene-specific roles of TF-binding sites, primarily because their roles often are not well understood. We introduce two models of regulatory sequence evolution that address some of the shortcomings of existing models and implement simulation frameworks based on them. One model simulates the evolution of an individual binding site in the context of a CRM, while the other evolves an entire CRM. Both models use a state-of-the art sequence-to-expression model to predict the effects of mutations on the regulatory output of the CRM and determine the strength of selection. We use the new framework to simulate the evolution of TF-binding sites in 37 well-studied CRMs belonging to the anterior-posterior patterning system in Drosophila embryos. We show that these simulations provide accurate fits to evolutionary data from 12 Drosophila genomes, which includes statistics of binding site conservation on relatively short evolutionary scales and site loss across larger divergence times. The new framework allows us, for the first time, to test hypotheses regarding the underlying cis-regulatory code by directly comparing the evolutionary implications of the hypothesis with the observed evolutionary dynamics of binding sites. Using this capability, we find that explicitly modeling self-cooperative DNA binding by the TF Caudal (CAD) provides significantly better fits than an otherwise identical evolutionary simulation that lacks this mechanistic aspect. This hypothesis is further supported by a statistical analysis of the distribution of intersite spacing between adjacent CAD sites. Experimental tests confirm direct homodimeric interaction between CAD molecules as well as self-cooperative DNA binding by CAD. We note that computational modeling of the D. melanogaster CRMs alone did not yield significant evidence to support CAD self-cooperativity. We thus demonstrate how specific mechanistic details encoded in CRMs can be revealed by modeling their evolution and fitting such models to multispecies data.

Dataset Information

Structure and dynamics of the second CARD of human RIG-I provide mechanistic insights into regulation of RIG-I activation.

Publications

Structure and dynamics of the second CARD of human RIG-I provide mechanistic insights into regulation of RIG-I activation.

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