Mn2+ Directly Activates cGAS and Structural Analysis Suggests Mn2+ Induces a Noncanonical Catalytic Synthesis of 2′3′-cGAMP

DNA binding allosterically activates the cytosolic DNA sensor cGAS (cyclic GMP-AMP [cGAMP] synthase) to synthesize 2'3'-cGAMP, using Mg²⁺ as the metal cofactor that catalyzes two nucleotidyl-transferring reactions. We previously found that Mn²⁺ potentiates cGAS activation, but the underlying mechanism remains unclear. Here, we report that Mn²⁺ directly activates cGAS. Structural analysis reveals that Mn²⁺-activated cGAS undergoes globally similar conformational changes to DNA-activated cGAS but forms a unique η1 helix to widen the catalytic pocket, allowing substrate entry and cGAMP synthesis. Strikingly, in Mn²⁺-activated cGAS, the linear intermediates pppGpG and pGpA take an inverted orientation in the active pocket, suggesting a noncanonical but accelerated cGAMP cyclization without substrate flip-over. Moreover, unlike the octahedral coordination around Mg²⁺, the two catalytic Mn²⁺ are coordinated by triphosphate moiety of the inverted substrate, independent of the catalytic triad residues. Our findings thus uncover Mn²⁺ as a cGAS activator that initiates noncanonical 2'3'-cGAMP synthesis.