Computational design of constitutively active cGAS

• Published in: Nature structural & molecular biology Vol. 30; no. 1; pp. 72 - 80
• Main Authors: Dowling, Quinton M., Volkman, Hannah E., Gray, Elizabeth E., Ovchinnikov, Sergey, Cambier, Stephanie, Bera, Asim K., Sankaran, Banumathi, Johnson, Max R., Bick, Matthew J., Kang, Alex, Stetson, Daniel B., King, Neil P.
• Format: Journal Article
• Language: English
• Published: New York Nature Publishing Group US 01-01-2023; Nature Publishing Group
• Subjects: 631/250; 631/45/607; 631/535/1266; 82; 82/83; Adjuvants; Biochemistry; Biological activity; Biological Microscopy; Biomedical and Life Sciences; Cell cycle; Computer applications; Crystallography; Cyclic GMP; Deoxyribonucleic acid; Design; DNA; DNA - chemistry; DNA damage; Enzymes; Genetic code; Immune response; Immunity, Innate; Inflammatory diseases; Informatics; Innate immunity; Interferon; Life Sciences; Ligands; Localization; Membrane Biology; Molecular biology; Mutation; Nucleotidyltransferases - metabolism; Pattern recognition; Pattern recognition receptors; Protein Structure; Proteins; Receptors; Senescence; Tumor cells; Tumorigenesis; Tumors; X-ray crystallography
• ISSN: 1545-9993; 1545-9985
• DOI: 10.1038/s41594-022-00862-z

Cyclic GMP–AMP synthase (cGAS) is a pattern recognition receptor critical for the innate immune response to intracellular pathogens, DNA damage, tumorigenesis and senescence. Binding to double-stranded DNA (dsDNA) induces conformational changes in cGAS that activate the enzyme to produce 2′-3′ cyclic GMP–AMP (cGAMP), a second messenger that initiates a potent interferon (IFN) response through its receptor, STING. Here, we combined two-state computational design with informatics-guided design to create constitutively active, dsDNA ligand-independent cGAS (CA-cGAS). We identified CA-cGAS mutants with IFN-stimulating activity approaching that of dsDNA-stimulated wild-type cGAS. DNA-independent adoption of the active conformation was directly confirmed by X-ray crystallography. In vivo expression of CA-cGAS in tumor cells resulted in STING-dependent tumor regression, demonstrating that the designed proteins have therapeutically relevant biological activity. Our work provides a general framework for stabilizing active conformations of enzymes and provides CA-cGAS variants that could be useful as genetically encoded adjuvants and tools for understanding inflammatory diseases.
The authors use computational protein design to stabilize the active conformation of cGAS, generating constitutively active cGAS variants that could potentiate prophylactic and therapeutic effects.