Figure 3
V-cGAPs specifically degrade 3′3′-cGAMP. (A) Four chemically synthesized cGAMP isoforms were incubated with purified VCA0681 for 2 h, and then the cGAMP activity assay was performed. (B) HPLC assay was performed after incubation of cGAMPs with the indicated purified V-cGAPs for 10 h. (C, D) 2.5 μg of 3′3′-cGAMP (C) or indicated amounts of 3′3′-cGAMP (D) was incubated with 3 μg of purified VCA0681 for the indicated times (C) or 10 h (D). The cGAMP activity assay was then performed. (E, F) VCA0681 HD/AA mutants as indicated were expressed and purified from E. coli(E, bottom), and incubated with 3′3′-cGAMP for 2 h (E) or 40 min (F). The cGAMP activity assay (E, top) or HPLC analysis (F) was then performed. M1: H74A/D75A; M2: H288A/D289A; DM: H74A/D75A/H288A/D289A; DM/WT: A74H/A75D/A288H/A289D. (G) Similar to E, purified VCA0681 mutants as indicated (bottom) were incubated with 3′3′-cGAMP for 2 h, and then the cGAMP activity assay (top) was performed. Y: Y350A; HH: H341A/H342A/E343A; GK: G291A/K292A; DG: D346A/G347A.
