Nicking mechanism underlying the DNA phosphorothioate-sensing antiphage defense by SspE

Abstract

DNA phosphorothioate (PT) modification, with a nonbridging phosphate oxygen substituted by sulfur, represents a widespread epigenetic marker in prokaryotes and provides protection against genetic parasites. In the PT-based defense system Ssp, SspABCD confers a single-stranded PT modification of host DNA in the 5′-CPSCA-3′ motif and SspE impedes phage propagation. SspE relies on PT modification in host DNA to exert antiphage activity. Here, structural and biochemical analyses reveal that SspE is preferentially recruited to PT sites mediated by the joint action of its N-terminal domain (NTD) hydrophobic cavity and C-terminal domain (CTD) DNA binding region. PT recognition enlarges the GTP-binding pocket, thereby increasing GTP hydrolysis activity, which subsequently triggers a conformational switch of SspE from a closed to an open state. The closed-to-open transition promotes the dissociation of SspE from self PT-DNA and turns on the DNA nicking nuclease activity of CTD, enabling SspE to accomplish self-nonself discrimination and limit phage predation, even when only a small fraction of modifiable consensus sequences is PT-protected in a bacterial genome.

Conserved domains can be found across distinct phage defence systems

“…SspE proteins, part of SspABCD-SspE phosphorothioationsensing bacterial defence systems, also contain predicted DUF262 and DUF1524 domains. Nevertheless, the reported role of SspE appears distinct from that observed for GrmSD and BrxU (Gao et al, 2022;Xiong et al, 2020). The SspE structure highlighted the DUF262 retains the conserved DGQQR motif in the nucleotidebinding pocket (Gao et al, 2022;Xiong et al, 2020).…”

“…Nevertheless, the reported role of SspE appears distinct from that observed for GrmSD and BrxU (Gao et al, 2022;Xiong et al, 2020). The SspE structure highlighted the DUF262 retains the conserved DGQQR motif in the nucleotidebinding pocket (Gao et al, 2022;Xiong et al, 2020). As per BrxU, SspE DUF262 can hydrolyse GTP, CTP, UTP and ATP indiscriminately.…”

“…Whereas DUF1524 of GmrSD and BrxU causes cleavage of modified DNA, within SspE the DUF1524 promotes DNA nicking (Gao et al, 2022;Xiong et al, 2020).…”

Vibrio choleraepathogenicity island 2 encodes two distinct types of restriction systems

“…Notably, the DGQQR motif is thought to form part of a nucleotide binding pocket and to be required for nucleotide hydrolysis. Indeed, site-directed mutants of either TgvA or TgvB encoding substitutions in this motif previously shown to disrupt NTPase activity (48–50), all resulted in a total loss of anti-phage activity (Fig. 4c-e).…”

“…Machnicka et al, showed that the predominant form of GmrSD is as a single multi-domain protein containing an N-terminal DUF262(GmrS) domain and a Cterminal DUF1524(GmrD) domain, separated by an alpha helical linker region (44). This domain organisation was subsequently confirmed by crystal structures of the related GmrSD family members BrxU, which also recognises and degrades DNA containing modified cytosines, and the phosphorothioate modification sensing enzyme SspE (48)(49)(50). Furthermore, biochemical experiments with these enzymes have shown that the N-terminal DUF262 likely functions as DNA modification sensor, and uses nucleotide binding and hydrolysis to regulate the activity of the C-terminal DUF1524, which functions as a nuclease to degrade non-self-DNA (48,50).…”