Structure, substrate binding and activity of a unique AAA plus protein: the BrxL phage restriction factor

Bacteriophage exclusion ('BREX') systems are multi-protein complexes encoded by a variety of bacteria and archaea that restrict phage by an unknown mechanism. One BREX factor, termed BrxL, has been noted to display sequence similarity to various AAA+ protein factors including Lon protease. In this study we describe multiple CryoEM structures of BrxL that demonstrate it to be a chambered, ATP-dependent DNA binding protein. The largest BrxL assemblage corresponds to a dimer of heptamers in the absence of bound DNA, versus a dimer of hexamers when DNA is bound in its central pore. The protein displays DNA-dependent ATPase activity, and ATP binding promotes assembly of the complex on DNA. Point mutations within several regions of the protein-DNA complex alter one or more in vitro behaviors and activities, including ATPase activity and ATP-dependent association with DNA. However, only the disruption of the ATPase active site fully eliminates phage restriction, indicating that other mutations can still complement BrxL function within the context of an otherwise intact BREX system. BrxL displays significant structural homology to MCM subunits (the replicative helicase in archaea and eukaryotes), implying that it and other BREX factors may collaborate to disrupt initiation of phage DNA replication.

Automated summary

Bacteriophage exclusion (BREX) systems, encoded by bacteria and archaea, limit phage infection via an unknown mechanism. One component, BrxL, shares sequence similarity with AAA+ proteins like Lon protease. CryoEM structures reveal BrxL as a chambered, ATP-dependent DNA binding protein. Mutations affecting ATPase activity disrupt various in vitro behaviors, but only mutations disrupting the ATPase active site completely eliminate phage restriction, suggesting possible collaboration between BrxL and other BREX factors in disrupting phage DNA replication initiation.