BioRxiv (2023) PubMed PMID: 37546940; PubMed Central PMCID: PMC10402079.

Abstract

The mammalian innate immune system uses cyclic GMP–AMP synthase (cGAS) to synthesize the cyclic dinucleotide 2′,3′-cGAMP during antiviral and antitumor immune responses. 2′,3′-cGAMP is a nucleotide second messenger that initiates inflammatory signaling by binding to and activating the stimulator of interferon genes (STING) receptor. Bacteria also encode cGAS/DncV-like nucleotidyltransferases (CD-NTases) that produce nucleotide second messengers to initiate antiviral (antiphage) signaling. Bacterial CD-NTases produce a wide range of cyclic oligonucleotides but have not been documented to produce 2′,3′-cGAMP. Here we discovered bacterial CD-NTases that produce 2′,3′-cGAMP to restrict phage replication. Bacterial 2′,3′-cGAMP binds to CD-NTase associated protein 14 (Cap14), a transmembrane protein of unknown function. Using electrophysiology, we show that Cap14 is a chloride-selective ion channel that is activated by 2′,3′-cGAMP binding. Cap14 adopts a modular architecture, with an N-terminal transmembrane domain and a C-terminal nucleotide-binding SAVED domain. Domain-swapping experiments demonstrated the Cap14 transmembrane region could be substituted with a nuclease, thereby generating a biosensor that is selective for 2′,3′-cGAMP. This study reveals that 2′,3′-cGAMP signaling extends beyond metazoa to bacteria. Further, our findings suggest that transmembrane proteins of unknown function in bacterial immune pathways may broadly function as nucleotide-gated ion channels.

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Citation

Tak U, Walth P, Whiteley AT. bioRxiv. 2023 Jul 24;. doi: 10.1101/2023.07.24.550367. PubMed PMID: 37546940; PubMed Central PMCID: PMC10402079.

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Methods Enzymol. 2019;625:13-40. doi: 10.1016/bs.mie.2019.04.012. Epub 2019 May 2.

Abstract

Cyclic GMP-AMP synthase (cGAS) is an innate immune system enzyme responsible for recognition of double-stranded DNA aberrantly localized in the cell cytosol. cGAS binds DNA and is activated to catalyze production of the nucleotide second messenger 2'-5'/3'-5' cyclic GMP-AMP (2'3' cGAMP). In spite of a major role for cGAS in the cellular immune response, a complete understanding of cGAS biology has been limited by a lack of genetic tools to rapidly screen cGAS activity, instability of human cGAS-DNA interactions in vitro, and a previous absence of structural information for the human cGAS-DNA complex. Here we detail procedures to map the molecular determinants of cGAS activation and describe methods developed to prepare human cGAS-DNA crystals for structural analysis. Together with earlier systems established to study mammalian homologs of cGAS, these innovations provide a foundation to understand and therapeutically target human cGAS biology.

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*co-corresponding authors

Nature. 2019 Mar;567(7747):194-199. doi: 10.1038/s41586-019-0953-5. Epub 2019 Feb 20.

Abstract

Cyclic dinucleotides (CDNs) have central roles in bacterial homeostasis and virulence by acting as nucleotide second messengers. Bacterial CDNs also elicit immune responses during infection when they are detected by pattern-recognition receptors in animal cells. Here we perform a systematic biochemical screen for bacterial signalling nucleotides and discover a large family of cGAS/DncV-like nucleotidyltransferases (CD-NTases) that use both purine and pyrimidine nucleotides to synthesize a diverse range of CDNs. A series of crystal structures establish CD-NTases as a structurally conserved family and reveal key contacts in the enzyme active-site lid that direct purine or pyrimidine selection. CD-NTase products are not restricted to CDNs and also include an unexpected class of cyclic trinucleotide compounds. Biochemical and cellular analyses of CD-NTase signalling nucleotides demonstrate that these cyclic di- and trinucleotides activate distinct host receptors and thus may modulate the interaction of both pathogens and commensal microbiota with their animal and plant hosts.

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• [Cell host & microbe 2019]
• [Science Signaling 2019]
• Highlighted and discussed in This Week In Microbiology Podcast 

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*These authors contributed equally to this work

Cell. 2018 Jul 12;174(2):300-311.e11. doi: 10.1016/j.cell.2018.06.026.

Abstract

Cyclic GMP-AMP synthase (cGAS) recognition of cytosolic DNA is critical for immune responses to pathogen replication, cellular stress, and cancer. Existing structures of the mouse cGAS-DNA complex provide a model for enzyme activation but do not explain why human cGAS exhibits severely reduced levels of cyclic GMP-AMP (cGAMP) synthesis compared to other mammals. Here, we discover that enhanced DNA-length specificity restrains human cGAS activation. Using reconstitution of cGAMP signaling in bacteria, we mapped the determinant of human cGAS regulation to two amino acid substitutions in the DNA-binding surface. Human-specific substitutions are necessary and sufficient to direct preferential detection of long DNA. Crystal structures reveal why removal of human substitutions relaxes DNA-length specificity and explain how human-specific DNA interactions favor cGAS oligomerization. These results define how DNA-sensing in humans adapted for enhanced specificity and provide a model of the active human cGAS-DNA complex to enable structure-guided design of cGAS therapeutics.

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•  [Immunity. 2018]

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