1. Volume 158, Issue 5, Pages 1011-1021 (August 2014)
Structure-Guided Reprogramming of Human cGAS Dinucleotide Linkage Specificity 
Philip J. Kranzusch, Amy S.Y. Lee, Stephen C. Wilson, Mikhail S. Solovykh, Russell E. Vance, James M. Berger, Jennifer A. Doudna 
Cell 
Volume 158, Issue 5, Pages (August 2014)
DOI: /j.cell
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2. Cell  , DOI: ( /j.cell )
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3. Figure 1
Structure of Vibrio cholerae DncV Reveals an Autoactivated cGAS-like Enzyme
(A) Crystal structure of Vibrio cholerae DncV. α helices are colored in green, β strands forming the enzyme core are in blue, and a red circle marks the active site.
(B) Superposition of the crystal structures of DncV (green) and human cGAS (purple) (PDB 4KM5).
(C) Surface view representation of the DncV (green), apo human cGAS (purple, “inactive”), and dsDNA-bound mouse cGAS (purple, “active”) active site substrate channels (mouse cGAS PDB 4K98). Nucleotide substrate binding pockets are colored in blue, and the active site is marked by a red circle.
(D) Superposition of an electrostatic surface view of DncV colored by charge (positive, blue and negative, red) and cGAS-bound dsDNA (green). The structures of DncV and dsDNA-bound mouse cGAS were superposed without manual fitting of the dsDNA ligand.
See also Figure S1.
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4. Figure 2 DncV GTP Recognition Parallels cGAS Substrate Discrimination
(A) Analysis of DncV and human cGAS cyclic dinucleotide product formation. Reactions were labeled with indicated ɑ-32P and unlabeled (“cold”) nucleotide substrate, and all cGAS reactions included dsDNA. Reactions were treated with alkaline phosphatase and separated by thin layer chromatography. The lower panel represents a longer exposure, and images are representative of multiple independent experiments.
(B) Crystal structure of DncV bound to Mg2+ ions (pink) and GTP analog (yellow/orange).
(C) Alignment of DncV (green) and cGAS (purple) substrate nucleotides derived from superposition of the DncV-GTP and pig cGAS-ATP/GTP (PDB 4KB6) crystal structures.
(D) Zoomed view of GTP analog bound in the DncV active site.
(E and F) Cartoon schematic of DncV interactions with the catalytic Mg2+ ions (metal A and metal B). Octahedral coordination was initially determined by building into the higher-resolution 1.8 Å maps of the catalytically trapped structure.
(G) Cartoon schematic of DncV interactions with the bound GTP analog as described in the Results.
See also Figure S3.
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5. Figure 3
Prokaryotic and Metazoan Dinucleotide Cyclization Reactions Proceed in Opposing Directions
(A) Crystal structure of DncV bound to Mg2+ ions (pink) and a linear pppA[3ʹ–5ʹ]pG intermediate (yellow/orange).
(B) Fo-Fc omit map of the pppA[3ʹ–5ʹ]pG ligand contoured to 4.5 σ.
(C) Cartoon schematic of DncV interactions with the pppA[3ʹ–5ʹ]pG as described in the Results.
(D) Analysis of DncV and human cGAS cyclic dinucleotide product formation as in Figure 2A. Reactions include labeled/unlabeled NTPs and nonhydrolyzable, methylene-substituted ATP and GTP nucleotides as indicated. A[3ʹ–5ʹ]pG (green) and G[2ʹ–5ʹ]pA (purple) labels denote migration of trapped linear products from DncV and cGAS respectively, and the image is representative of multiple independent experiments.
(E) Reaction schematic of DncV and cGAS dinucleotide product formation.
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6. Figure 4
Active Site Remodeling Controls Substrate Rotation and Phosphodiester Linkage Specificity
(A and B) Zoomed view of the substrate binding pocket in the pppA[3ʹ–5ʹ]pG bound DncV structure (green) and pppG[2ʹ–5ʹ]pG bound mouse cGAS structure (purple).
(C and D) Cartoon schematic illustrating site 1, 2, and 3 positions leading to altered substrate base rotation in DncV (green) and cGAS (purple).
(E) Superposition of bound linear intermediate substrates from DncV (green) and cGAS (purple) illustrating an ∼40° rotation in nucleotide base positioning.
(F) Schematic illustrating substrate rotation model where elongating amino acids in site 1 or site 2 reposition prokaryotic (green) and eukaryotic (purple) bases, respectively.
(G) WebLogo illustration of site 1, 2, and 3 conservation in prokaryotic (green) and eukaryotic (purple) cGAS-like enzyme active sites. Text size correlates with conservation across alignment of all available DncV and cGAS sequences (Crooks et al., 2004).
See also Figures S1 and S2 and Table S2.
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7. Figure 5 Rational Reprogramming of Human cGAS Product Formation
(A) Analysis of DncV and human cGAS cyclic dinucleotide product formation as in Figure 2A. Reactions include DncV, WT cGAS, or the indicated cGAS mutant enzymes, and migration of A[3ʹ–5ʹ]pG[3ʹ–5ʹ]p (DncV, green label) and WT G[2ʹ–5ʹ]pA[3ʹ–5ʹ]p (cGAS, purple label) products are indicated. MBP-tagged WT cGAS produces more linear G[2ʹ–5ʹ]pA than untagged protein (see Figure 2A).
(B) High-resolution mass spectrometry analysis of cGAS R376I product formation confirms production of cyclic AMP-GMP, as well as cdA and linear intermediates, as discussed in the Results.
(C) Dinucleotide analysis as in A, with WT cGAS or the indicated cGAS mutant enzymes in the presence of absence of dsDNA stimulation.
(D) Dinucleotide analysis as in A, with alkaline phosphate-treated products additionally digested with Nuclease P1 to hydrolyze all 3ʹ–5ʹ linkages (right lanes). WT cGAS G[2ʹ–5ʹ]pA[3ʹ–5ʹ]p product is digested by Nuclease P1 to the indicated G[2ʹ–5ʹ]pA intermediate, whereas DncV and reprogrammed cGAS products are digested to completion. Lower panels display increased exposures of the same experiments as in Figure 2, and images are representative of multiple independent experiments.
See also Figures S3, S4, and S5.
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8. Figure 6
Reprogrammed Human cGAS Enzymes Signal Discrete Alleles of the Adaptor STING
(A) In-cell reconstitution of cyclic dinucleotide immune activation. Cells were cotransfected with empty vector, human cGAS, or Vibrio DncV plasmids in the presence of WT mouse STING (mSTING WT) or the R232H allele of human STING incapable of responding to 3ʹ–5ʹ cGAMP (hSTING R232H). Activating dsDNA was cotransfected in all samples, and immune activation was monitored with a firefly luciferase reporter under control of the IFN-β promoter (pIFNβ-FLuc).
(B) Cells were transfected as in A and included reprogrammed human cGAS enzymes as indicated. Data are normalized to the empty vector control, and error bars represent the SD from the mean of at least three independent experiments (the asterisk denotes p < 0.002, and n.s. denotes not significant).
See also Figure S5.
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9. Figure S1
Vibrio DncV Amino Acid Sequence Conservation, Related to Figures 1 and 4
(A) Alignment of DncV amino-acid sequences generated with MAAFT and colored by BLOSUM62 conservation score (see methods). Cartoon schematic of the experimentally determined Vibrio DncV secondary structure is depicted below (green = ɑ-helix, blue = β strand) and red dots denote the conserved active site residues involved in metal coordination. Regions with no observable electron density in the DncV crystal structure (F144–K149 and F218–T238) are faded.
(B) Structural alignment of Vibrio DncV and human cGAS sequences based on superposition of pppA[3ʹ–5ʹ]pG and cGAS pppG[2ʹ–5ʹ]pG (PDB 4KM5 and 4K98) structures (see methods). Alignment is colored as in A, and experimentally determined secondary-structures are depicted for DncV above the sequence alignment (green = ɑ-helix, blue = β strand) and for cGAS below the sequence alignment (purple = ɑ-helix, blue = β strand).
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10. Figure S2 Vibrio DncV Crystal Contacts, Related to Figure 1
(A) Packing of individual DncV monomers in the crystallographic asymmetric unit. A red circle denotes the packing region between chain A (green/blue) and chain B (gray/blue), and the calculated buried surface area is indicated. Bound Mg2+ ions (pink) and pppA[3ʹ–5ʹ]pG ligand (yellow/orange) are depicted as spheres.
(B) Packing of individual DncV monomers in adjacent symmetry-related molecules. DncV chain A monomer is displayed as in A, and the symmetry-related molecule is depicted as a faded out monomer. Packing regions and calculated buried surface area are indicated in red as in A.
(C) Vibrio DncV size-exclusion chromatography purification data demonstrating DncV migrates as a monomer during purification according to its predicted molecular weight. Migration of standards are indicated in gray (IgG ∼158 kDa, Albumin ∼67 kDa, Ovalbumin ∼43 kDa, Myoglobin ∼17 kDa).
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11. Figure S3
DncV Activity Is Insensitive to Nucleic Acid, Related to Figures 2 and 5
(A) Analysis of DncV and human cGAS cyclic dinucleotide product formation as in Figure 3. Prior to substrate nucleotide addition, DncV reactions were supplemented with 20 nt ssRNA, 45 nt ssDNA or 45 bp dsDNA as indicated (see methods); these substrates have no apparent impact on product A[3ʹ–5ʹ]pG[3ʹ–5ʹ]p (cAG) formation.
(B) DncV reactions prepared as in A, but with 5ʹ radiolabeled nucleic acid substrates and separated by non-denaturing polyacrylamide gel-electrophoresis. Radiolabeled substrates include 20 nt ssRNA, 20 bp dsRNA, ∼56 bp structured human 5ʹ Jun UTR mRNA, 45 nt ssDNA, 45 bp dsDNA or a branched ss/dsDNA substrate as previously described to stimulate Bacillus subtilis DisA (Witte et al., 2008). Control reactions include corresponding radiolabeled substrate in the absence of purified DncV (left lanes).
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12. Figure S4
Reprogrammed cGAS Enzymes Have Reduced Guanine Selectivity, Related to Figure 5
Analysis of DncV, human cGAS and reprogrammed cGAS cyclic dinucleotide formation as in Figure 3D. Reprogrammed cGAS enzymes have a reduced selectivity for order of guanine nucleotide selection but preferentially proceed in the same direction as WT cGAS. Additionally, consistent with relaxed guanine-specific base interactions, reprogrammed cGAS mutants produce cyclic di-A (A[3ʹ–5ʹ]pA[3ʹ–5ʹ]p) under low GTP conditions, as described in the main text. Reactions include labeled/unlabeled NTPs and nonhydrolyzable methylene-substituted ATP and GTP nucleotides as indicated. A[3ʹ–5ʹ]pG (green), G[3ʹ–5ʹ]pA (green) and G[2ʹ–5ʹ]pA (purple) labels denote migration of trapped linear products from DncV and cGAS respectively, and images are representative of multiple independent experiments.
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13. Figure S5 DncV Reprogramming Mutations, Related to Figures 5 and 6
In-cell reconstitution of cyclic dinucleotide immune activation by Vibrio DncV as in Figure 6. Cells were co-transfected with indicated empty vector, human cGAS or Vibrio DncV plasmids in the presence of WT STING or the R232H allele of STING incapable of responding to 3ʹ–5ʹ cGAMP. Immune activation was monitored with a firefly luciferase reporter under control of the IFN-beta promoter (pIFNβ-FLuc). Data are normalized to WT human cGAS signal as in Figure 6 and error bars represent the SD from the mean of at least three independent experiments (∗ denotes p < 0.002, and n.s. denotes not significant).
Cell  , DOI: ( /j.cell )
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