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Proc Natl Acad Sci U S A. 2019 Jun 11;116(24):11946-11955. doi: 10.1073/pnas.1905013116. Epub 2019 May 29.

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

Author information

1
Structural Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY 10065; xiew@mskcc.org pateld@mskcc.org.
2
Laboratory for RNA Molecular Biology, The Rockefeller University, New York, NY 10065.
3
High-Throughput and Spectroscopy Resource Center, The Rockefeller University, New York, NY 10065.
4
Tri-Institutional Therapeutics Discovery Institute, New York, NY 10021.

Abstract

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.

KEYWORDS:

DNA-binding cGAS mutations; h-cGAS–DNA complex; liquid-phase condensation; multivalent interactions

PMID:
31142647
PMCID:
PMC6575157
[Available on 2019-11-29]
DOI:
10.1073/pnas.1905013116

Conflict of interest statement

The authors declare no conflict of interest.

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