Structures and mechanism of E2-CBASS anti-phage system

Area of Science:

• Microbiology
• Molecular Biology
• Structural Biology

Background:

• Bacteria possess innate immune systems, including cyclic-oligonucleotide-based anti-phage signaling systems (CBASS), to defend against bacteriophage infections.
• CBASS systems utilize cGAS/DncV-like nucleotidyltransferases (CD-NTases) to synthesize cyclic-oligonucleotides, activating effectors that eliminate infected cells and prevent phage propagation.
• A significant class of CBASS involves ubiquitin-conjugating enzymes (E1-E2 fusion or E2) that regulate CD-NTase activity, yet structural data on these complexes remain limited.

Purpose of the Study:

• To analyze the length and classification of CD-NTases in type II CBASS systems, specifically E1E2/JAB-CBASS and E2-CBASS.
• To elucidate the structural basis of the interaction between CD-NTase and E2 components in the E2-CBASS system.
• To provide mechanistic insights into the function of the E2-CBASS system in bacterial antiviral defense.

Main Methods:

• Comparative analysis of CD-NTase length and phylogenetic classification across different CBASS types.
• X-ray crystallography to determine the structure of the SmCdnG-SmE2 complex bound to GTP.
• Structural analysis focusing on conserved domains and unique interaction interfaces between CD-NTase and E2 proteins.

Main Results:

• The CD-NTase in E2-CBASS systems is generally longer and primarily belongs to clade G compared to other CBASS types.
• The crystal structure of the SmCdnG-SmE2 complex reveals a conserved GTP binding pattern, essential for enzyme activity.
• A unique C-terminal α-helix and β-sheet structure in SmCdnG was identified as critical for complex formation with SmE2, while the E2 protein structure is highly conserved within the E2-CBASS system.

Conclusions:

• The study reveals distinct characteristics of CD-NTases in E2-CBASS systems, suggesting specialized roles in bacterial immunity.
• Structural data of the SmCdnG-SmE2 complex provides a mechanistic understanding of how E2 regulates CD-NTase activity.
• These findings enhance our comprehension of prokaryotic antiviral defense mechanisms and the structural diversity within CBASS systems.