The cytosolic DNA sensor cyclic GMP-AMP synthase (cGAS) mediates innate immune responses against invading pathogens, or against self-dsDNA, which causes autoimmune disorders. Upon nonspecific binding of cytosolic B–form DNA, cGAS synthesizes the second messenger 2′3′-cGAMP and triggers STING-dependent signaling to produce type I IFNs. The cGAS comprises less-conserved N-terminal residues and highly conserved nucleotidyltransferase/Mab21 domains. The function and structure of the well-conserved domains have been extensively studied, whereas the physiological function of the N-terminal domain of cGAS is largely uncharacterized. In this study we used a single-molecule technique combined with traditional biochemical and cellular assays to demonstrate that binding of nonspecific dsDNA by the N-terminal domain of cGAS promotes its activation. We have observed that the N terminus of human cGAS (hcGAS-N160) undergoes secondary structural change upon dsDNA binding in solution. Furthermore, we showed that the hcGAS-N160 helps full length hcGAS to expand the binding range on λDNA and facilitates its binding efficiency to dsDNA compared with hcGAS without the 160 N-terminal residues (hcGAS-d160). More importantly, hcGAS-N160 endows full length hcGAS relatively higher enzyme activity and stronger activation of STING/IRF3-mediated cytosolic DNA signaling. These findings strongly indicate that the N-terminal domain of cGAS plays an important role in enhancing its function.
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This work is supported by the National Natural Science Foundation of China (31670740, U1430237, 31270803, and 31230023) and the Chinese Ministry of Science and Technology (2015CC040097 and 2014CB542600 to Z.J., 2014CB910102 to X.-D.S.).
The online version of this article contains supplemental material.
Abbreviations used in this article:
- circular dichroism
- cyclic GMP-AMP synthase
- fluorescein amidite
- human cGAS
- hcGAS without the 160 N-terminal residues
- full length hcGAS
- N terminus of human cGAS
- IFN-stimulatory DNA
- mouse cGAS
- microscale thermophoresis
- size-exclusion chromatography with multiangle light scattering
- Sendai virus.
- Received November 9, 2016.
- Accepted February 22, 2017.
- Copyright © 2017 by The American Association of Immunologists, Inc.