# Structures and mechanism of E2‐CBASS anti‐phage system

## TL;DR

This study explores the structure and function of the E2-CBASS system, a bacterial immune mechanism that fights phage infections without needing E1 enzymes.

## Contribution

The study reveals the structural and mechanistic details of the E2-CBASS system, including a conserved complex formed by CD-N-Tase and E2.

## Key findings

- The CD-N-Tase in E2-CBASS is longer and primarily belongs to clade G.
- The SmCdnG-SmE2 complex structure shows a conserved donor binding pattern and a unique C-terminal α-helix and β-sheet involved in complex formation.
- The E2 protein in E2-CBASS is highly conserved, suggesting a critical role in the system's function.

## Abstract

Bacteria deploy diverse innate immune systems to combat bacteriophage infections. The cyclic‐oligonucleotide‐based anti‐phage signaling system (CBASS) is a type of innate prokaryotic immune system. CBASS synthesizes cyclic‐oligonucleotide through cGAS/DncV‐like nucleotidyltransferases (CD‐NTases) to activate downstream effectors, which kill bacteriophage‐infected bacteria, thereby stopping phage spread. One major class of CBASS contains a homolog of eukaryotic ubiquitin‐conjugating enzymes, either as an E1‐E2 fusion or a single E2 enzyme. Both enzymes function by regulating CD‐NTase activity. Currently, many structures of CD‐NTases have been reported, but there are only a few reports of structures where CD‐NTases form complexes with the associated E2. In this study, we analyzed the length and classification of the CD‐NTase in two types of type II CBASS—E1E2/JAB‐CBASS and E2‐CBASS. We found that the CD‐NTase in E2‐CBASS is longer and predominantly belongs to clade G. We also present the structure of the SmCdnG‐SmE2 complex with the bound GTP substrate, which indicates the conservation of the donor binding pattern. Interestingly, we discovered that SmCdnG contains a conserved C‐terminal α‐helix and β‐sheet structure, which is uniquely involved in forming a complex with SmE2. We also found that the structure of the E2 protein in the E2‐CBASS system is highly conserved. Altogether, we provide mechanistic insights into the E2‐CBASS system.

Cyclic oligonucleotide‐based anti‐phage signaling system (CBASS) is an important component of prokaryotic innate immunity. This study reveals the structural basis and regulatory mechanism of the E2‐CBASS system, a unique pathway that operates without E1 homologs. Through cryo‐EM analysis and functional validation, we identify critical interactions between CD‐NTase and E2 and uncover a distinct mode of complex assembly. These findings provide valuable insights into CBASS defense systems and expand our understanding of the diversity of prokaryotic immune mechanisms.