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

Abstract

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.

Molecular basis of foreign DNA recognition by BREX anti-phage immunity system

“…BrxC may play a role of the assembling scaffold/recruitment unit for the complex, similar to the role of ORC, which loads the replicative helicase MCM onto replication origins 66,67 . Strikingly, BrxL protein shares a distinct homology with the MCM helicases 38 and thus could represent a tentative BREX translocase. Supporting this hypothesis, Type II, III, V, and VI BREX systems that lack BrxL encode BrxH helicase instead.…”

“…Other core BREX proteins include BrxZ (PglZ) belonging to alkaline phosphatase superfamily and homologous to the phosphodiesterase PorX 35 , and BrxC (PglY), an AAA+ ATPase with distant homology to ORC/Cdc6 (Origin Recognition Complex) proteins 36,37 . In addition, Type I BREX systems encode a DNA-binding AAA+ ATPase BrxL 38 , and two small proteins of unknown function BrxA 39 and BrxB ( Figure 1A ). Type I BREX gene clusters are often associated with genes coding for additional accessory proteins, for example, the WYL-domain transcriptional regulator BrxR 33,40 , which is also found in other defense systems such as CBASS 41 .…”

Structure and rational engineering of the PglX methyltransferase and specificity factor for BREX phage defence

“…3a ). BrxL was recently shown to form a dimer of hexameric rings, forming a barrel-like structure that binds and translocates along DNA 24 . Thus, BrxL had been considered to have an essential role as the “effector” for BREX phage defence.…”

“…There are six BREX sub-types, and type I BREX contains six genes; brxA , brxB , brxC , pglX , pglZ and brxL 10 . BrxA is a DNA-binding protein 23 , and BrxL is a DNA-stimulated AAA+ ATPase 24 . PglX has sequence and structural homology to methyltransferases and is hypothesised to methylate non-palindromic 6 bp sequences (BREX motifs) on the N6 adenine at the fifth position of the motif 10,22,25 , allowing discrimination between self and non-self DNA.…”

The vibriophage-encoded inhibitor OrbA abrogates BREX-mediated defense through the ATPase BrxC

“…BrxL was previously predicted to have a Lon-like domain; however, a recent 3D structure of BrxL from Acinetobacter sp. 394 solved by cryo-electron microscopy shows BrxL has structural similarity to the MCM replicative helicase from archaea and eukaryotes 22 . How the BREX components function together to modulate modification and restriction is poorly understood.…”