Mn2+ Directly Activates cGAS and Triggers A Distinct 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, utilizing 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 reported that Mn2+ directly activated cGAS. Structural analysis revealed that Mn2+-activated cGAS underwent globally similar conformational changes to DNA-activated cGAS, but formed 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 took an inverted orientation in the active pocket, indicating a noncanonical but accelerated cGAMP cyclization without substrate flip-over. Moreover, unlike the octahedral coordination around Mg2+, the two catalytic Mn2+ were coordinated by triphosphate moiety of the inverted substrate independent of the catalytic triad residues. Our findings thus uncovered Mn2+ as a cGAS activator that initiates a distinct 2’3’-cGAMP synthesis.