Structural characterization of anti-CRISPR protein AcrIE9.

The arms race between bacteria and bacteriophages has driven the evolution of both CRISPR-Cas systems and anti-CRISPR (Acr) proteins. AcrIE9, a type I-E Acr protein identified in Pseudomonas aeruginosa, inhibits Cascade-mediated DNA binding by interacting with the Cas7e subunit. However, its structural basis and precise inhibitory mechanism have remained unclear. Here, we report the crystal structure of AcrIE9 at 1.73 Å resolution, along with additional structural and biochemical analyses. AcrIE9 exists as both monomer and dimer in solution, while the crystal structure reveals a homodimeric assembly. Each protomer adopts a unique α/β architecture, and structural similarity searches indicate that AcrIE9 represents a previously uncharacterized protein fold. In vitro binding assays using individually purified type I-E Cas subunits from P. aeruginosa did not detect direct interaction with AcrIE9, including with Cas7e. These findings suggest that AcrIE9 may recognize a composite interface formed only within the intact Cascade complex, consistent with the AlphaFold3 prediction of multivalent interactions with Cas7e subunits. Taken together, this study provides the structural characterization of AcrIE9 and supports an inhibitory mechanism involving a multi-subunit binding surface on Cascade.