Topological rearrangements activate the HerA-DUF anti-phage defense system

Leveraging the rich structural information provided by AlphaFold, we used integrated experimental approaches to characterize the HerA-DUF4297 (DUF) anti-phage defense system, in which DUF is of unknown function. To infer the function of DUF, we performed structure-guided genomic analysis and found that DUF homologs are universally present in bacterial immune defense systems. One notable homolog of DUF is Cap4, a universal effector with nuclease activity in CBASS, the most prevalent anti-phage system in bacteria. To test the inferred nuclease function of DUF, we performed biochemical experiments and discovered that the DUF only exhibits activity against DNA substrates when it is bound by HerA. To understand how HerA activates DUF, we determined the structures of DUF and the HerA-DUF complex. DUF forms large oligomeric assemblies with or without HerA, suggesting that oligomerization per se is not sufficient for DUF activation. Instead, DUF activation requires dramatic topological rearrangements that propagate from HerA to the entire HerA-DUF complex, leading to reorganization of DUF for effective DNA cleavage. We further validated these structural insights by structure- guided mutagenesis. Together, these findings reveal dramatic topological rearrangements throughout the HerA-DUF complex, challenge the long-standing dogma that protein oligomerization alone activates immune signaling, and may inform the activation mechanism of CBASS.