Structures and mechanism of E2-CBASS anti-phage system

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.

Keywords: CBASS; CD‐NTase; anti‐phage defense system; cryo‐EM structure; ubiquitin.

Figure 1

Statistics of CD‐NTase in two type II CBASS systems and the covalent linkage between SmCdnG and SmE2. (A) Length of CD‐NTase in E1E2/JAB‐CBASS and E2‐CBASS systems. The results show that CD‐NTases in E2‐CBASS are longer than those in E1E2/JAB‐CBASS. (B) Classification of CD‐NTase in two type II CBASS systems. In the E2‐CBASS system, CD‐NTases are mainly concentrated in clade G. (C) SDS‐PAGE analysis showing the in vitro formation of the SmCdnG–SmE2 complex. (D) The C‐terminal sequence of the SmCdnG protein modified by inserting a TEV site and Strep‐Tag II between G397 and G398. (E, F) SDS‐PAGE (E) and Western blot (F) analyses showing that the SmCdnG‐TEV‐Strep‐SmE2 was cut by TEV.

Figure 2

Cryo‐EM structure of the SmCdnG‐SmE2 complex with GTP. (A) The cryo‐EM map of the SmCdnG–SmE2 complex with GTP, overlaid with a cartoon representation of the model created with ChimeraX. SmCdnG‐SmE2 is a heterodimer, with SmCdnG binding one molecule of GTP. (B) Overview of the SmCdnG‐SmE2 interfaces, with the covalent linkage between SmCdnG and SmE2 highlighted by a red dashed line. The SmCdnG‐SmE2 complex also has three additional interaction interfaces. (C–E) Detailed view of interacting residues at interface 1 (C), interface 2 (D), and interface 3 (E) between SmCdnG and SmE2. Black dashed lines represent hydrogen bonds. (F) SDS‐PAGE analysis of SmCdnG–SmE2 complex formation in wild‐type and interface‐mutant variants.

Figure 3

Structural and sequence analysis of SmCdnG with GTP. (A) Structure of SmCdnG with bound GTP. (B) Alignment of the structure of SmCdnG with bound GTP and the apo SmCdnG structure. The N‐lobe of SmCdnG with bound GTP is tilted by 3.4°. (C) Detailed view of the GTP binding site in SmCdnG. Multiple conserved residues interact with GTP. (D) Structure‐guide alignment of different CdnG subclasses, with the catalytic site sequences highlighted, performed using ESPript 3.2.

Figure 4

Structural and sequence analysis of CD‐NTase from different E2‐CBASS systems. (A) The extra α‐helix and β‐sheet in the C‐terminus of the SmCdnG. (B) Superposition of truncated BdCdnG structure (PDB ID: 7LJN) and AlphaFold 3 predicted full‐length BdCdnG structure. The extra α‐helix and β‐sheet in the C‐terminus of the BdCdnG. (C) Protein structure of Clade H protein GjCdnH in E2‐CBASS predicted by AlphaFold 3. (D) Structure guide alignment of CD‐NTase C‐terminal region from different E2‐CBASS systems by ESPript 3.2.

Figure 5

Structural and sequence analysis of E2 proteins from different E2‐CBASS systems. (A) Superposition of SmE2 structure used in this study and human E2 structure (PDB ID: 4AUQ). (B) Structure of GjE2 of E2‐CBASS predicted by AlphaFold 3. (C) Structure of BdE2 of E2‐CBASS predicted by AlphaFold 3. (D) Structure‐guide alignment of E2 by ESPript 3.2.