0428同步年報-2021-全

048 ACTIVITY REPORT 2021 reversed disposition, unlikely to react with the donor. Nevertheless, this binding mode is consistent with the ITC results that also showed two sites for ATP. Occupation of the acceptor site by a reversed ATP molecule might be a result of its large concentration in crystallization. Inhibition by ATP might serve as a regulation mechanism of Ec CdnD, a Type-II CD-NTase. When ATP is abundant, indicating a thriving condition, suicide might not be necessary. ATP at a large concentration was also shown to inhibit mammalian cGAS, 7 despite its being a Type-I enzyme. Structural comparison with DncV indicates that the “cage” of Ec CdnD is more open at the acceptor end, at which a positively charged surface patch is seen in a groove between the βF-βG and α8-α9 loops (the “gateway”). It corresponds to the binding site for the triphosphate of the “fake acceptor”, and might allow extension of the real intermediate farther from the active site. Upon superposition with DncV and Ec CdnD, the primer strand in RNA polymerase penetrates the “cage” of DncV but fits roughly into the “gateway” of Ec CdnD, whereas the P-site and A-site nucleotides match well with the acceptor and donor substrates. Taken together, the CTN-producing path of Ec CdnD might begin with two consecutive RNA Fig. 2 : Ligand-binding modes in the Ec CdnD crystals. The bound nucleotides are shown as thick sticks and the amino-acid residues as thin sticks. Mg 2+ ions and water molecules are shown as purple and red spheres. Hydrogen bonds and coordination bonds are indicated with dashed lines. The models of the bound ligands are superimposed on Fo-Fc omit maps contoured at 4σ level. (a) The bound ddATP in the tetragonal crystal has an associated Mg 2+ ion. (b) At the C-terminus of helix α7 a structural Mg 2+ ion is bound. (c) The donor ATP in the monoclinic crystal shows interactions via the ribose 2'- and 3'-OH groups and a second Mg 2+ ion. (d) In the orthorhombic crystal the acceptor nucleotide AMPcPP interacts with three arginines and a third Mg 2+ ion. [Reproduced from Ref. 5] Fig. 3 : Shared mechanism of the NTase-catalyzed reaction. The Mg 2+ ion bound to the acceptor ribose is designated metal A; that bound to the donor triphosphate is metal B. The nucleophilic attack of O3' (or O2') at 5'-phosphate is indicated with an arrow. [Reproduced from Ref. 5] (a) (b) (c) (d)