The appearance of DNA in the cytoplasm is a very dangerous signal to cells. This could be caused by viral or bacterial infection, or as a result of cellular damage that leaks host DNA into the cytoplasm. Cytosolic DNA activates the host immune response, which is critically dependent on the newly identified cGAS-cGAMP-STING pathway. Structure-function studies from our laboratories have demonstrated 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]. Further, cyclization occurs in a stepwise manner through initial generation of 5'-pppG(2',5')A prior to cyclization to c[G(2',5')pA(3',5')p], with the latter positioned precisely in the catalytic pocket. These studies established c[G(2',5')pA(3',5')p] as a founding member of a family of metazoan 2',5'-containing cyclic heteronucleotide second messengers distinct from bacterial 3',5' cyclic dinucleotides. Follow up structure-function studies from our laboratories have shown that human and mouse STING undergo an ‘open’ to ‘closed’ conformational transition upon binding c[G(2',5')pA(3',5')p] and the anti-viral agent DMXAA. Comparing hSTING to mSTING in cellular assays, 2',5'-linkage-containing cGAMP isomers were more specific triggers of the IFN pathway compared to the all 3',5'-linkage isomer counterparts. Our results highlight the critical role of the lid residue at position 230 (Gly in hSTING; Ile in mSTING) for the DMXAA species selectivity. Our structural and functional results also shed light on strategies to restore an efficient DMXAA-response of hSTING based on the binding pocket S162A and Q266I substitutions.
Citation Format: Pu Gao, Manuel Ascano, Thomas Zillinger, Winfried Barchet, Thomas Tuschl, Dinshaw J. Patel. Structure-function studies of cytosolic DNA sensing pathway. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B030.