Spectroscopic characterization of Mn²⁺-induced catalysis in cyclic GMP-AMP synthase

Cyclic GMP-AMP synthase (cGAS) is a nucleotidyltransferase enzyme that functions as the principal cytosolic sensor of double-stranded DNA (dsDNA) in vertebrates. Upon binding dsDNA, cGAS produces the second messenger 2'3'-cyclic GMP-AMP (2'3'-cGAMP), initiating innate immune signaling. Canonical models describe activation through dsDNA-induced dimerization and recruitment of two Mg²⁺ ions into the active site. Recent studies, however, have identified Mn²⁺ as an alternative cofactor that accelerates catalysis, enhances substrate selectivity, and improves product fidelity. Interestingly, in the presence of dsDNA, Mn²⁺ acts as the preferred catalytic metal, and in its absence, it can promote dsDNA-independent synthesis of 2'3'-cGAMP. The metal-dependent divergence in activity raises key questions about the composition of the active metal cluster and the molecular basis for catalytic enhancement. Here, liquid chromatography-tandem mass spectrometry (LC-MS/MS) and electron paramagnetic resonance (EPR) spectroscopy, including electron spin echo envelope modulation (ESEEM) and electron-nuclear double resonance (ENDOR), were used to probe Mn²⁺ coordination and its interaction with substrates. Mn²⁺-cGAS generated approximately sixfold more cGAMP than Mg²⁺-cGAS with dsDNA and fourteenfold more without dsDNA, confirming enhanced catalysis and dsDNA-independent activation. CW and pulsed EPR revealed that Mn²⁺ uptake occurs during active-site assembly and that Mn²⁺ is coordinated by ATP and protein-derived ligands in the substrate-bound complex. These results provide the first spectroscopic description of Mn²⁺ coordination in cGAS and offer critical mechanistic insight into its metal-dependent activation and broader role in health and disease. Additionally, this work further defines how metal cofactors regulate innate immune signaling by cGAS.