Structural characterization of Class 2 OLD family nucleases supports a two-metal catalysis mechanism for cleavage

Abstract

Overcoming lysogenization defect (OLD) proteins constitute a family of uncharacterized nucleases present in bacteria, archaea, and some viruses. These enzymes contain an N-terminal ATPase domain and a C-terminal Toprim domain common amongst replication, recombination, and repair proteins. The in vivo activities of OLD proteins remain poorly understood and no definitive structural information exists. Here we identify and define two classes of OLD proteins based on differences in gene neighborhood and amino acid sequence conservation and present the crystal structures of the catalytic C-terminal regions from the Burkholderia pseudomallei and Xanthamonas campestris p.v. campestris Class 2 OLD proteins at 2.24 Å and 1.86 Å resolution respectively. The structures reveal a two-domain architecture containing a Toprim domain with altered architecture and a unique helical domain. Conserved side chains contributed by both domains coordinate two bound magnesium ions in the active site of B. pseudomallei OLD in a geometry that supports a two-metal catalysis mechanism for cleavage. The spatial organization of these domains additionally suggests a novel mode of DNA binding that is distinct from other Toprim containing proteins. Together, these findings define the fundamental structural properties of the OLD family catalytic core and the underlying mechanism controlling nuclease activity.

A nucleotide-sensing endonuclease from the Gabija bacterial defense system

“…Based on sequence homology, the OLD family proteins are the closest to GajA among characterized nucleases ( Figure S11). BpOLD and TsOLD also consist of an ATPase domain and a TOPRIM domain, resembling the domain organization of GajA (19,20). As previously reported, the ATPase domain of TsOLD is functional in ATP hydrolysis and the TOPRIM domain carries out non-specific nuclease activity (20).…”

“…The Gabija system exists in about 8.5% of all sequenced bacteria and archaea (10). In most cases it consists of two components, GajA and GajB, while in some cases the GajB predicted as a helicase is absent (10,19,20). Bioinformatics analysis indicated that GajA contains an ATPase-like domain (residues 1-341) and a TOPRIM domain (residues 348-510) ( Figure 1A).…”

“…In this study, we aim to elucidate the function of GajA protein, which consists of an Nterminal ATPase-like domain and a C-terminal TOPRIM domain and was predicted as an ATP-dependent nuclease. Among characterized nucleases, the recently reported nonspecific nucleases of the OLD family involved in DNA repair and replication, including BpOLD, XccOLD and TsOLD (19,20), share the highest homology with GajA. GajB was predicted as a UvrD helicase and in some bacterial genomes the GajB homolog is absent thus the GajA homolog is the sole component of potential Gabija (10,19,20).…”

Genome sequence of Pseudomonas aeruginosa PAO1161, a PAO1 derivative with the ICEPae1161 integrative and conjugative element

“…The effect of a mutation is predicted using the PAO1-UW genome as a reference. In case of PA2492 (mexT) the nucleotide changes are proposed to alter the start codon and hence the sequence of N-terminal part (OLD) family nuclease containing an N-terminal ATPase domain and a C-terminal TOPRIM domain [52,53]. Since OLD proteins can act as exonucleases digesting DNA in the 5′-3′ direction as well as endonucleases acting on supercoiled, circular DNA substrates [53], it was tempting to speculate that PA1939 could play a role in degradation of the foreign DNA in concert with PA2735 acting as methylase.…”

Structures of B. subtilis Maturation RNases Captured on 50S Ribosome with Pre-rRNAs

“…Structural comparison of the M5 NTD with the Burkholderia pseudomallei OLD (BpOLD) protein (PDB 6NK8), which cleaves one strand of a DNA duplex using a catalytic Toprim domain (Schiltz et al, 2019), reveals a common spatial organisation of active-site acidic residues ( Figure 5B). Like BpOLD, M5 has a strictly conserved Toprim catalytic triad E10/D56/D58 (D58 here mutated to A), and additional conserved, acidic residues (D14, E96), that are positioned for coordination of two Mg 2+ ions ( Figure 5B).…”