Figure 1.
Crystal structure of human cGAS.
(A) Overall architecture. The model is shown as a ribbon representation. The α-helices and β-sheets of the N-terminal core are colored violet and pink, respectively. The zinc finger and the C-terminal bundle are colored gold and cyan, respectively. The bound zinc ion is shown as a grey sphere. Secondary structures are numbered. (B) The active site. The catalytic triad residues (Glu225, Asp227 and Asp319) are shown as ball-and-stick models. (C) The zinc finger. The zinc-coordinating residues are shown as ball-and-stick models.
Figure 2.
Structural rearrangements of cGAS.
(A) Structural comparisons of our human apo-cGAS with the previously determined human (left; PDB 4KM5), mouse (middle; PDB 4K8V) and porcine (right; PDB 4JLX) apo-cGASs. Our human cGAS is shown as a ribbon model, with the same coloring as in Figure 1A. The other cGASs are shown as silver ribbon models. (B) Close-up view of α1 in human apo (left), mouse apo and its complex with 18 bp dsDNA (PDB 4K96) (middle), and porcine apo and its complex with 14 bp dsDNA (PDB 4KB6) (right). The mouse and porcine apo-cGASs are shown as silver ribbon models, and the cGASs complexed with dsDNAs are shown as orange ribbon models. dsDNAs are shown as brown ribbons. The leucine residue on α1 and the catalytic triad are shown as ball-and-stick models.
Figure 3.
(A) Electrostatic surface potential of human cGAS. (B) (C) Mouse (B) and porcine (C) cGAS complexes with dsDNA. The models are shown as ribbon representations.
Figure 4.
Identification of crucial residues of human cGAS for IRF3 and NF-B activation.
(A) Human cGAS mutants used for functional analyses. Human cGAS is shown as a ribbon model, with the same coloring as in Figure 1A. Mutated residues are shown as red ball-and-stick models. (B) cGAS-induced phosphorylation of TBK1, IRF3, and STING. cGAS WT or mutants were expressed in HEK293T cells stably expressing human STING. Cell lysates were analyzed by western blotting, using the indicated antibodies. The asterisk indicates phosphorylated STING. (C) Reporter assays for IFN-β (left panel) and NF-κB (right panel). The cell lysates are the same as in (B), except for the co-expression with reporter plasmids, and were measured for luciferase activities. Luciferase activities are shown as mean ± s.d. (n = 3). (D) Induction of IFN-β and A20 by human cGAS and its mutants. The relative mRNA expression levels of IFN-β (left) and A20 (right) were analyzed by Real-Time PCR using total RNAs isolated from the cells shown in (B). Relative expression levels are shown as mean ± s.d. (n = 3). (E) Immunoblotting for cGAS-induced phosphorylation (left panel), reporter assays for IFN-β (upper panel) and NF-κB (lower panel), the same as (B) and (C), respectively. (F) Pull-down experiment between biotinylated-ISD and human cGAS mutants. Human cGAS mutants were expressed and purified from E. coli, and mixed with Streptavidin beads in the presence or absence of biotinylated-ISD. Bound proteins were eluted with SDS sample buffer and analyzed by SDS-PAGE.