title: Structure-guided reprogramming of human cGAS dinucleotide linkage specificity.
abstract: Cyclic dinucleotides (CDNs) play central roles in bacterial pathogenesis and innate immunity. The mammalian enzyme cGAS synthesizes a unique cyclic dinucleotide (cGAMP) containing a 2'-5' phosphodiester linkage essential for optimal immune stimulation, but the molecular basis for linkage specificity is unknown. Here, we show that the Vibrio cholerae pathogenicity factor DncV is a prokaryotic cGAS-like enzyme whose activity provides a mechanistic rationale for the unique ability of cGAS to produce 2'-5' cGAMP. Three high-resolution crystal structures show that DncV and human cGAS generate CDNs in sequential reactions that proceed in opposing directions. We explain 2' and 3' linkage specificity and test this model by reprogramming the human cGAS active site to produce 3'-5' cGAMP, leading to selective stimulation of alternative STING adaptor alleles in cells. These results demonstrate mechanistic homology between bacterial signaling and mammalian innate immunity and explain how active site configuration controls linkage chemistry for pathway-specific signaling.
authors.affiliation: Department of Molecular & Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA; Howard Hughes Medical Institute (HHMI), University of California, Berkeley, Berkeley, CA 94720, USA.
authors.forename: Philip J
authors.initials: PJ
authors.lastname: Kranzusch
authors.affiliation: Department of Molecular & Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA; Center for RNA Systems Biology, University of California, Berkeley, Berkeley, CA 94720, USA.
authors.forename: Amy S Y
authors.initials: ASY
authors.lastname: Lee
authors.affiliation: Department of Chemistry, University of California, Berkeley, Berkeley, CA 94720, USA.
authors.forename: Stephen C
authors.initials: SC
authors.lastname: Wilson
authors.affiliation: Department of Molecular & Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA.
authors.forename: Mikhail S
authors.initials: MS
authors.lastname: Solovykh
authors.affiliation: Department of Molecular & Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA; Howard Hughes Medical Institute (HHMI), University of California, Berkeley, Berkeley, CA 94720, USA.
authors.forename: Russell E
authors.initials: RE
authors.lastname: Vance
authors.affiliation: Department of Biophysics, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA. Electronic address: jmberger@jhmi.edu.
authors.forename: James M
authors.initials: JM
authors.lastname: Berger
authors.affiliation: Department of Molecular & Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA; Center for RNA Systems Biology, University of California, Berkeley, Berkeley, CA 94720, USA; Howard Hughes Medical Institute (HHMI), University of California, Berkeley, Berkeley, CA 94720, USA; Department of Chemistry, University of California, Berkeley, Berkeley, CA 94720, USA; Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA. Electronic address: doudna@berkeley.edu.
authors.forename: Jennifer A
authors.initials: JA
authors.lastname: Doudna
mesh_terms: D000595:Amino Acid Sequence; D020134:Catalytic Domain; D006801:Humans; D007113:Immunity, Innate; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009713:Nucleotidyltransferases / Q000737:chemistry* / Q000378:metabolism; D015202:Protein Engineering; D016415:Sequence Alignment; D013379:Substrate Specificity; D014734:Vibrio cholerae / Q000201:enzymology*; D000099235:Cyclic Guanosine Monophosphate-Adenosine Monophosphate Synthase
chemical_list: D009713:Nucleotidyltransferases; C578390:cGAS protein, human; D000099235:Cyclic Guanosine Monophosphate-Adenosine Monophosphate Synthase