Human cGAS catalytic domain has an additional DNA-binding interface that enhances enzymatic activity and liquid-phase condensation

The cyclic GMP-AMP synthase (cGAS)–cGAMP–STING pathway plays a key role in innate immunity, with cGAS sensing both pathogenic and mislocalized DNA in the cytoplasm. Human cGAS (h-cGAS) constitutes an important drug target for control of antiinflammatory responses that can contribute to the onset of...

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Veröffentlicht in:Proceedings of the National Academy of Sciences - PNAS 2019-06, Vol.116 (24), p.11946-11955
Hauptverfasser: Xie, Wei, Lama, Lodoe, Adura, Carolina, Tomita, Daisuke, Glickman, J. Fraser, Tuschl, Thomas, Patel, Dinshaw J.
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container_end_page 11955
container_issue 24
container_start_page 11946
container_title Proceedings of the National Academy of Sciences - PNAS
container_volume 116
creator Xie, Wei
Lama, Lodoe
Adura, Carolina
Tomita, Daisuke
Glickman, J. Fraser
Tuschl, Thomas
Patel, Dinshaw J.
description The cyclic GMP-AMP synthase (cGAS)–cGAMP–STING pathway plays a key role in innate immunity, with cGAS sensing both pathogenic and mislocalized DNA in the cytoplasm. Human cGAS (h-cGAS) constitutes an important drug target for control of antiinflammatory responses that can contribute to the onset of autoimmune diseases. Recent studies have established that the positively charged N-terminal segment of cGAS contributes to enhancement of cGAS enzymatic activity as a result of DNA-induced liquid-phase condensation. We have identified an additional cGASCD–DNA interface (labeled site-C; CD, catalytic domain) in the crystal structure of a human SRY.cGASCD–DNA complex, with mutations along this basic site-C cGAS interface disrupting liquid-phase condensation, as monitored by cGAMP formation, gel shift, spin-down, and turbidity assays, as well as time-lapse imaging of liquid droplet formation. We expand on an earlier ladder model of cGAS dimers bound to a pair of parallel-aligned DNAs to propose a multivalent interaction-mediated cluster model to account for DNA-mediated condensation involving both the N-terminal domain of cGAS and the site-C cGAS–DNA interface. We also report the crystal structure of the h-cGASCD–DNA complex containing a triple mutant that disrupts the site-C interface, with this complex serving as a future platform for guiding cGAS inhibitor development at the DNA-bound h-cGAS level. Finally, we solved the structure of RU.521 bound in two alternate alignments to apo h-cGASCD, thereby occupying more of the catalytic pocket and providing insights into further optimization of active-site–binding inhibitors.
doi_str_mv 10.1073/pnas.1905013116
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Recent studies have established that the positively charged N-terminal segment of cGAS contributes to enhancement of cGAS enzymatic activity as a result of DNA-induced liquid-phase condensation. We have identified an additional cGASCD–DNA interface (labeled site-C; CD, catalytic domain) in the crystal structure of a human SRY.cGASCD–DNA complex, with mutations along this basic site-C cGAS interface disrupting liquid-phase condensation, as monitored by cGAMP formation, gel shift, spin-down, and turbidity assays, as well as time-lapse imaging of liquid droplet formation. We expand on an earlier ladder model of cGAS dimers bound to a pair of parallel-aligned DNAs to propose a multivalent interaction-mediated cluster model to account for DNA-mediated condensation involving both the N-terminal domain of cGAS and the site-C cGAS–DNA interface. We also report the crystal structure of the h-cGASCD–DNA complex containing a triple mutant that disrupts the site-C interface, with this complex serving as a future platform for guiding cGAS inhibitor development at the DNA-bound h-cGAS level. Finally, we solved the structure of RU.521 bound in two alternate alignments to apo h-cGASCD, thereby occupying more of the catalytic pocket and providing insights into further optimization of active-site–binding inhibitors.</abstract><cop>United States</cop><pub>National Academy of Sciences</pub><pmid>31142647</pmid><doi>10.1073/pnas.1905013116</doi><tpages>10</tpages><orcidid>https://orcid.org/0000-0002-4550-6211</orcidid><oa>free_for_read</oa></addata></record>
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subjects Amino Acid Sequence
AMP
Autoimmune diseases
Binding
Biological Sciences
Catalysis
Catalytic Domain - physiology
Condensation
Crystal structure
Cyclic GMP
Cytoplasm
Deoxyribonucleic acid
Dimers
Disruption
DNA
DNA - metabolism
DNA structure
Enzymatic activity
Humans
Immunity
Immunity, Innate - physiology
Innate immunity
Liquid phases
Membrane Proteins - metabolism
Mutation
Nucleotides, Cyclic - metabolism
Nucleotidyltransferases - metabolism
Optimization
PNAS Plus
Sequence Alignment
Signal Transduction - physiology
Turbidity
title Human cGAS catalytic domain has an additional DNA-binding interface that enhances enzymatic activity and liquid-phase condensation
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