Structural insights into distinct filamentation states reveal a regulatory mechanism for bacterial STING activation

The cyclic oligonucleotide-based antiphage signaling system (CBASS) is a bacterial immune mechanism that was evolutionarily linked to the eukaryotic cGAS–STING pathway, which protects against phage infection through abortive cell death. CBASS operons encode cyclic dinucleotide synthases (CD-NTases) and effector proteins (Caps), such as bacterial STING, which senses cyclic dinucleotides like 3′3′-c-di-GMP to trigger defense. Although bacterial STING oligomerizes into filaments upon ligand binding, the functional roles of distinct filament states remain unclear. Here, we resolve cryo-EM structures of Epilithonimonas lactis TIR-STING (ElSTING) bound to 3′3′-c-di-GMP, revealing two oligomeric states: spiral-shaped single filaments and fiber bundles composed of straight protofibrils. In spiral filaments, the STING domain sequesters the TIR domain’s BB loop within a hydrophobic core, suppressing NADase activity. This inactive conformation is stabilized by interactions between the CBDα4 helix and the TIR domain, as well as a calcium-binding site. Conversely, fiber bundle formation—driven by inter-protofibril TIR domain interactions—disrupts these autoinhibitory contacts, liberating the BB loop to enable head-to-tail assembly of adjacent TIR domains into a composite NADase-active site. Calcium ions promote spiral filament assembly while inhibiting fiber bundles, revealing a dual regulatory role in tuning ElSTING activation. Strikingly, this mechanism diverges from single-filament systems like SfSTING, underscoring evolutionary diversity in STING signaling. Our findings establish distinct filamentfilamentfilamentarchitectures as structural checkpoints governing bacterial STING activation, providing mechanistic insights into how conformational plasticity and environmental cues like calcium regulate abortive infection. These results highlight parallels between prokaryotic and eukaryotic immune strategies, emphasizing conserved principles in pathogen defense across domains of life.