Anti-CRISPR (Acr) proteins are natural inhibitors of CRISPR-Cas systems, found in bacteriophages and other genetic elements. AcrIE3, identified in a Pseudomonas phage, inactivates the type I-E CRISPR-Cas system in Pseudomonas aeruginosa by engaging with the Cascade complex. However, its precise inhibition mechanism has remained elusive. In this study, we present a comprehensive structural and biochemical analysis of AcrIE3, providing mechanistic insight into its anti-CRISPR function. Our results reveal that AcrIE3 selectively binds to the Cas8e subunit of the Cascade complex. The crystal structure of AcrIE3 exhibits an all-helical fold with a negatively charged surface. Through extensive mutational analyses, we show that AcrIE3 interacts with the protospacer adjacent motif (PAM) recognition site in Cas8e through its negatively charged surface residues. These findings enhance our understanding of the structure and function of type I-E Acr proteins, suggesting PAM interaction sites as primary targets for divergent Acr inhibitors.
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