ATP-dependent dimerization of BrxC coordinates BrxBCZ assembly for phage exclusion

Abstract Bacteriophage Exclusion (BREX) systems protect bacterial genomes via sequence-specific DNA methylation while blocking phage replication through a poorly understood “restriction-without-cleavage” mechanism. Here, we present cryo-EM structures and functional characterization of the core BREX machinery from Escherichia coli . We determined the structures of the ATPase BrxC in its monomeric state and two ATPγS-bound dimeric conformations, showing that ATP binding, not hydrolysis, triggers dimerization. The conserved Arg328 couples dimer formation to ATP hydrolysis and is required for both phage restriction and host methylation; the R328A mutant supports assembly but abolishes defense. We further resolved the BrxBCZ ternary complex, revealing BrxB as a central scaffold bridging the BrxC dimer and BrxZ without direct BrxC–BrxZ contact. Biochemical assays show that BrxZ is a dual-function enzyme with alkaline phosphatase and metal-dependent nuclease activities. Thus, ATP drives BrxC assembly and inhibits BrxZ activities. Disruption of the BrxB–BrxC interface or the BrxC ATPase cycle selectively eliminates restriction while preserving methylation, establishing BrxBCZ as a restriction-specific structural core. Collectively, these findings delineate the structural basis for ATP-dependent coordination of BREX, revealing how energy metabolism is integrated into bacterial innate immunity.