There is currently considerable interest towards gaining an improved molecular and functional understanding of innate immune sensors of higher metazoans that recognize nucleic acids in the cytoplasm and trigger type I interferon induction. Cytoplasmic dsDNA of pathogenic bacterial or viral origin, and perhaps also displaced nuclear or mitochondrial DNA following cellular stress, represent such a trigger. These events involving self-nucleic acid recognition, in turn, could trigger autoimmune diseases.
Pattern Recognition Receptors of dsDNA
Recently, cyclic GMP-AMP synthase (cGAS) was identified by the Zhijian Chen laboratory (University of Texas Southwestern Medical School) as a cytoplasmic DNA sensor that activates the type I interferon pathway by synthesizing the second messenger cGAMP. cGAS was shown to be a member of of the nucleotidyltransferase family and to be capable of generating cGAMP in vitro from GTP and ATP in the presence of dsDNA. Additional experiments involving either overexpression or knockdown of cGAS established that cGAMP bound to and activated STING, resulting in the activation of transcription factor IRF3 and subsequent induction of interferon beta. A challenge was to identify the phosphodiester linkages that characterized cGAMP, elucidate the molecular interactions associated with STING activation by cGAMP, as well as the structural basis underlying targeting of STING by anti-viral small molecules.
Binding pocket and lid region substitutions render human STING sensitive to mouse-selective drug DMXAA
The drug DMXAA (5,6-dimethylxanthenone-4-acetic acid) showed therapeutic promise against solid tumors in mouse models but subsequently failed in human clinical trials. DMXAA was later discovered to activate mouse but not human STING, an adaptor protein in the cyclic dinucleotide cGAMP-mediated signaling pathway, inducing type-I interferon expression. To facilitate the development of compounds that target human STING, we combined structural, biophysical and cellular assays as part of a multidisciplinary effort [Winfred Barchet (University Hospital-Bonn, Germany), Thomas Tuschl (Rockefeller, NY) and Liang Deng (MSKCC, NY)] to study mouse and human chimeric proteins and their interaction with DMXAA. We identified a single substitution (G230I) that enables a DMXAA-induced conformational transition of hSTING from an inactive ‘open’ to an active ‘closed’ state. We also identified a substitution within the binding-pocket (Q266I) that cooperates with G230I and the previously identified S162A binding-pocket point substitution, rendering hSTING highly sensitive to DMXAA. These findings should facilitate the reciprocal engineering of DMXAA analogs that bind and stimulate wild-type hSTING, and their exploitation for vaccine-adjuvant and anti-cancer drug development.
Gao, P., Zillinger, T., Wang, W., Ascano, M., Dai, P., Hartmann, G., Tuschl, T., Deng, L., Barchet, W. and Patel, D. J. (2014). Binding pocket and lid region substituents render human STING sensitive to mouse-selective drug DMXAA. Cell Reports 8, 1668-1676.
Structure-Function Analysis of STING Activation by c[G(2’,5’)pA(3’,5’)p] and Targeting by Anti-Viral DMXAA
Binding of dsDNA by cyclic GMP-AMP (cGAMP) synthase (cGAS) triggers formation of the metazoan second messenger c[G(2’,5’)pA(3’,5’)p], which binds the signaling protein STING with subsequent activation of the IFN pathway. Our structural studies establish that human hSTINGH232 undergoes a conformational change upon binding c[G(2’,5’)pA(3’,5’)p] and its linkage isomer c[G(2’,5’)pA(2’,5’)p], as does mouse mStingR231 on binding c[G(2’,5’)pA(3’,5’)p], c[G(3’,5’)pA(3’,5’)p] and the anti-viral agent DMXAA, leading to similar ‘closed’ conformations. Comparing hSTING to mSting, the laboratories of our collaborators Thomas Tuschl (Rockefeller University), Liang Ding (Memorial Sloan Kettering Cancer Center) and Winfried Barchet and Gunter Hartmann (University Hospital-Bonn) established that 2’,5’-linkage-containing cGAMP isomers were more specific triggers of the IFN pathway compared to the all-3’,5’-linkage isomer. Guided by structural information, the laboratories of our collaborators Winfried Barchet and Gunter Hartmann (University Hospital-Bonn) identified a unique point mutation (S162A) placed within the cyclic-dinucleotide-binding site of hSTING that rendered it sensitive to the otherwise mouse-specific drug DMXAA. Our structural and functional analysis highlights the unexpected versatility of STING in the recognition of natural and synthetic ligands involving multiple amino acids within a small-molecule pocket created by the dimerization of STING.
Gao, P., Ascano, M., Zillinger, T., Wang, Y., Dai, P., Serganov, A. A., Gaffney, B. L., Shuman, S., Jones, R., Deng, L., Hartmann, G., Barchet, W., Tuschl, T. and Patel, D.J. (2013). Structure-function studies of STING activation by c[G(2’,5’)pA(3’,5’)p], its linkage isomers and DMXAA. Cell 154, 748-762.
Cyclic [G(2’,5’)pA(3’,5’)p] is the Metazoan Second Messenger Produced by DNA-Activated Cyclic GMP-AMP Synthase
Recent studies have identified cyclic GMP-AMP (cGAMP) as a metazoan second messenger triggering an interferon response. cGAMP is generated from GTP and ATP by cytoplasmic dsDNA sensor cGAMP synthase (cGAS). We combined structural, chemical (laboratory of collaborator Roger Jones), biochemical (laboratory of collaborator Thomas Tuschl) and cellular (laboratories of collaborators Winfried Barchet and Gunter Hartmann) assays to demonstrate that this second messenger contains G(2’,5’)pA and A(3’,5’)pG phosphodiester linkages, designated c[G(2’,5’)pA(3’,5’)p]. Our structural studies demonstrate that upon dsDNA binding cGAS is activated through conformational transitions, resulting in formation of a catalytically competent and accessible nucleotide binding pocket for generation of c[G(2’,5’)pA(3’,5’)p]. We demonstrate that cyclization occurs in a stepwise manner through initial generation of 5’-pppG(2’,5’)pA prior to cyclization to c[G(2’,5’)pA(3’,5’)p], with the latter positioned precisely in the catalytic pocket. Mutants of cGAS dsDNA-binding or catalytic pocket residues exhibit reduced or abrogated activity. Our studies have identified c[G(2’,5’)pA(3’,5’)p] as a founding member of a new family of metazoan 2’,5’-containing cyclic hetero dinucleotide second messengers distinct from bacterial 3’,5’ cyclic dinucleotides.
Gao, P., Ascano, M., Wu, Y., Barchet, W., Gaffney, B. L., Zillinger, T., Serganov, A., Jones, R. A., Hartmann, G., Tuschl, T. and Patel, D. J. (2013). Cyclic [G(2’,5’)pA(3’,5’)p] is the metazoan second messenger produced by DNA-activated cyclic GMP-AMP synthase. Cell 153, 1094-1107.
Pattern Recognition Receptors of 5’-Triphosphorylated RNAs
Nucleic acid-sensing pattern recognition receptors come in several flavors, including toll-like receptors (TLRs), retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs) and nucleotide oligomerization domain (NOD)-like receptors (NLRs). The RLR class is localized to the cytoplasm with RIG-I specifically targeting 5’-triphosphate-containing viral RNAs, thereby distinguishing viral from host transcripts.
RIG-I is a cytosolic helicase that senses 5’-ppp-RNA contained in negative strand RNA viruses and triggers innate antiviral immune responses. Calorimetric binding studies establish that the RIG-I C-terminal regulatory domain (CTD) binds to blunt-end double-stranded 5’-ppp-RNA a factor of 17-fold more tightly than to its single-stranded counterpart. Here we report on the crystal structure of RIG-I CTD domain bound to both blunt-ends of a self-complementary 5’-ppp-dsRNA 12-mer, with interactions involving 5’-pp clearly visible in the complex. The structure, supported by mutation studies undertaken in the laboratory of our collaborator Thomas Tuschl (Rockefeller University) defines how a lysine-rich basic cleft within the RIG-I CTD domain sequesters the observable 5’-pp of the bound RNA, with a stacked Phe capping the terminal base pair. Key intermolecular interactions observed in the crystalline state are retained in the complex of 5’-ppp-dsRNA 24-mer and full-length RIG-I under in vivo conditions, as evaluated by the laboratory of our collaborator Gunter Hartmann (University Hospital-Bonn) from the impact of binding pocket RIG-I mutations and 2’-OCH3 RNA modifications on the interferon response.
Wang, Y., Ludwig, J., Schuberth, C., Goldeck, M., Schlee, M., Li, H., Juranek, S., Sheng, G., Micura, R., Tuschl, T., Hartmann, G. & Patel, D. J. (2010). Structural and functional insights into 5’-ppp-RNA pattern recognition by the innate immune receptor RIG-I. Nat. Struct. Mol. Biol. 17, 781-787.