Cyclase-generated 3',5'-cGMP activates PucsarTIR antiphage defense through a stepwise zig-zag aligned filament assembly

Authors

Chakravarti, A., Wu, Y., Burroughs, A. M., Zhang, Z., Fatma, S., Huang, R. H., Aravind, L., Nobrega, F. L., Patel, D. J.

Abstract

Bacteria use nucleotide second messengers to couple phage recognition to antiviral effector activation. Here, we identify Pucsar, an ofshoot of the Pycsar-related antiphage family, whose cyclases generate 3',5'-cGMP from GTP, that are associated with Toll/Interleukin-1 receptor (TIR) and transmembrane (TM) effectors. In the PucsarTIR system, cGMP produced by cyclase PucC, selectively activates the cognate PucTIR NADase. X-ray crystallography shows that PucC adopts a dimeric cyclase scaffold with a guanine-compatible base-recognition pocket. Cryo-EM and mutational analyses revealed that apo-PucTIR, composed of cyclic nucleotide binding and TIR domains, is self-sequestered as an inactive tetramer, in which TIR domains are proximal but misoriented. Sequential cGMP binding remodels this assembly through partially occupied intermediates and stabilizes a fully cGMP-bound dimer, that polymerizes into a zig-zag aligned filament, creating composite NAD+-binding pockets required for efficient NADase activity. These findings establish 3',5'-cGMP as an antiphage second messenger and reveal how cyclic purine signaling converts self-sequestered TIR effectors into a NADase-competent filament.

Preprint server: bioRxiv
The authors list and abstract were imported from bioRxiv on 15 Aug 2026.

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