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RNA binding activates RIG-I by releasing an autorepressed signaling domain.

ABSTRACT: The retinoic acid-inducible gene I (RIG-I) innate immune receptor is an important immunotherapeutic target, but we lack approaches for monitoring the physical basis for its activation in vitro. This gap in our understanding has led to confusion about mechanisms of RIG-I activation and difficulty discovering agonists and antagonists. We therefore created a novel fluorescence resonance energy transfer-based method for measuring RIG-I activation in vitro using dual site-specific fluorescent labeling of the protein. This approach enables us to measure the conformational change that releases the signaling domain during the first step of RIG-I activation, making it possible to understand the role of stimulatory ligands. We have found that RNA alone is sufficient to eject the signaling domain, ejection is reversible, and adenosine triphosphate plays but a minor role in this process. These findings help explain RIG-I dysfunction in autoimmune disease, and they inform the design of therapeutics targeting RIG-I.

SUBMITTER: Dickey TH

PROVIDER: S-EPMC6774723 | biostudies-literature | 2019 Oct

REPOSITORIES: biostudies-literature

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RNA binding activates RIG-I by releasing an autorepressed signaling domain.

Dickey T H TH Song B B Pyle A M AM

Science advances 20191002 10

The retinoic acid-inducible gene I (RIG-I) innate immune receptor is an important immunotherapeutic target, but we lack approaches for monitoring the physical basis for its activation in vitro. This gap in our understanding has led to confusion about mechanisms of RIG-I activation and difficulty discovering agonists and antagonists. We therefore created a novel fluorescence resonance energy transfer-based method for measuring RIG-I activation in vitro using dual site-specific fluorescent labelin ...[more]

PMID: 31616790

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Project description:Key pointsThe electrogenic Na+ /HCO3 - cotransporter NBCe1-B is widely expressed in many tissues, including pancreas, submandibular gland, brain, heart, etc. NBCe1-B has very low activity under basal condition due to auto-inhibition, but can be fully activated by protein interaction with the IP3R-binding protein released with inositol 1,4,5-trisphosphate (IRBIT). The structural components of the auto-inhibition domain and the IRBIT-binding domain of NBCe1-B are finely characterized based on systematic mutations in the present study and data from previous studies. Reducing negative charges on the cytosol side of the transmembrane domain greatly decreases the magnitude of the auto-inhibition of NBCe1-B. We propose that the auto-inhibition domain functions as a brake module that inactivates NBCe1-B by binding to, via electrostatic attraction, the transmembrane domain; IRBIT activates NBCe1-B by releasing the brake from the transmembrane domain via competitive binding to the auto-inhibition domain.AbstractThe electrogenic Na+ /HCO3 - cotransporter NBCe1-B is widely expressed in many tissues in the body. NBCe1-B exhibits only basal activity due to the action of the auto-inhibition domain (AID) in its unique amino-terminus. However, NBCe1-B can be activated by interaction with the IP3R-binding protein released with inositol 1,4,5-trisphosphate (IRBIT). Here, we investigate the molecular mechanism underlying the auto-inhibition of NBCe1-B and its activation by IRBIT. The IRBIT-binding domain (IBD) of NBCe1-B spans residues 1-52, essentially consisting of two arms, one negatively charged (residues 1-24) and the other positively charged (residues 40-52). The AID mainly spans residues 40-85, overlapping with the IBD in the positively charged arm. The magnitude of auto-inhibition of NBCe1-B is greatly decreased by manipulating the positively charged residues in the AID or by replacing a set of negatively charged residues with neutral ones in the transmembrane domain. The interaction between IRBIT and NBCe1-B is abolished by mutating a set of negatively charged Asp/Glu residues (to Asn/Gln) plus a set of Ser/Thr residues (to Ala) in the PEST domain of IRBIT. However, this interaction is not affected by replacing the same set of Ser/Thr residues in the PEST domain with Asp. We propose that: (1) the AID, acting as a brake, binds to the transmembrane domain via electrostatic interaction to slow down NBCe1-B; (2) IRBIT activates NBCe1-B by releasing the brake from the transmembrane domain.

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RNA binding activates RIG-I by releasing an autorepressed signaling domain.

Publications

RNA binding activates RIG-I by releasing an autorepressed signaling domain.

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