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bioRxiv · 10.64898/2026.08.12.744502

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

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

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Chakravarti, A., Wu, Y., Burroughs, A. M., Zhang, Z., Fatma, S., Huang, R. H., Aravind, L., Nobrega, F. L., Patel, D. J.. 2026-08-17. Cyclase-generated 3,5-cGMP activates PucsarTIR antiphage defense through a stepwise zig-zag aligned filament assembly. https://doi.org/10.64898/2026.08.12.744502

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