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

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


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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Catalytic Domain / physiology*
  • DNA / metabolism*
  • Humans
  • Immunity, Innate / physiology
  • Membrane Proteins / metabolism
  • Nucleotides, Cyclic / metabolism
  • Nucleotidyltransferases / metabolism*
  • Sequence Alignment
  • Signal Transduction / physiology


  • Membrane Proteins
  • Nucleotides, Cyclic
  • cyclic guanosine monophosphate-adenosine monophosphate
  • DNA
  • Nucleotidyltransferases
  • cGAS protein, human

Associated data

  • PDB/6EDB
  • PDB/6EDC
  • PDB/6O47