期刊
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
卷 116, 期 24, 页码 11946-11955出版社
NATL ACAD SCIENCES
DOI: 10.1073/pnas.1905013116
关键词
h-cGAS-DNA complex; DNA-binding cGAS mutations; multivalent interactions; liquid-phase condensation
资金
- Tri-Institutional Therapeutics Discovery Institute (TDI)
- Takeda Pharmaceutical Company
- TDI's parent institutes (MSKCC, The Rockefeller University, and Weill Cornell Medicine)
- William H. Goodwin and Alice Goodwin from the Commonwealth Foundation for Research
- Center for Experimental Therapeutics of the MSKCC [GM104962, CA179564]
- MSKCC Core Grant [P30 CA008748]
- National Institute of General Medical Sciences [P41 GM103403]
- US Department of Energy [DE-AC02-06CH11357]
- Leona M. and Harry B. Helmsley Charitable Trust
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 cGAS(CD)-DNA interface (labeled site-C; CD, catalytic domain) in the crystal structure of a human SRY.cGAS(CD)-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-cGAS(CD)-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-cGAS(CD), thereby occupying more of the catalytic pocket and providing insights into further optimization of active-site-binding inhibitors.
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