cGAS activates innate immune responses against cytosolic double-stranded DNA. Here, by determining crystal structures of cGAS at various reaction stages, we report a unifying catalytic mechanism. apo-cGAS assumes an array of inactive conformations and binds NTPs nonproductively. Dimerization-coupled double-stranded DNA-binding then affixes the active site into a rigid lock for productive metal•substrate binding. A web-like network of protein•NTP, intra-NTP, and inter-NTP interactions ensures the stepwise synthesis of 2′−5′/3′−5′-linked cGAMP while discriminating against noncognate NTPs and off-pathway intermediates. One divalent metal is sufficient for productive substrate binding, and capturing the second divalent metal is tightly coupled to nucleotide and linkage specificities, a process which manganese is preferred over magnesium by 100-fold. Additionally, we elucidate how mouse cGAS achieves more stringent NTP and linkage specificities than human cGAS. Together, our results reveal that an adaptable, yet precise lock-and-key-like mechanism underpins cGAS catalysis.

cGAS 激活针对胞质双链 DNA 的先天免疫反应。在这里，通过确定 cGAS 在各个反应阶段的晶体结构，我们报告了统一的催化机制。 apo-cGAS 呈现一系列非活性构象并非生产性地结合 NTP。然后，二聚偶联的双链 DNA 结合将活性位点固定到刚性锁中，以实现高效的金属•底物结合。蛋白质·NTP、NTP内和NTP间相互作用的网状网络确保了2′−5′/3′−5′连接的cGAMP的逐步合成，同时区分非同源NTP和非途径中间体。一种二价金属足以有效地结合底物，并且捕获第二种二价金属与核苷酸和连接特异性紧密偶联，在这一过程中，锰比镁好 100 倍。此外，我们还阐明了小鼠 cGAS 如何实现比人类 cGAS 更严格的 NTP 和连锁特异性。总之，我们的结果表明，一种适应性强且精确的类似锁钥匙的机制支撑着 cGAS 催化。