Anti-CRISPR protein

AcrIE3 is a Type I-E anti-CRISPR (Acr) protein found in Pseudomonas bacteriophages. It functions as a potent inhibitor of the host bacterial CRISPR-Cas immune system, specifically targeting the Type I-E Cascade complex in Pseudomonas aeruginosa. By neutralizing the bacterial defense system, AcrIE3 allows the phage to replicate and survive within the host. ^[1]

Function

AcrIE3 functions through a mechanism of DNA mimicry to inhibit the CRISPR-Cas system.

• Target: The protein selectively binds to the Cas8e subunit of the multisubunit Cascade complex (Csy complex).
• Mechanism: AcrIE3 occupies the PAM (Protospacer Adjacent Motif) recognition site on Cas8e. By binding to this critical region, it sterically blocks the Cascade complex from recognizing and binding to the viral DNA target. This prevents the recruitment of the Cas3 nuclease and the subsequent degradation of the phage genome.
• Stoichiometry: The interaction typically involves one AcrIE3 molecule binding to one Cascade complex (1:1 stoichiometry with the Cas8e subunit).

Relevance

The discovery and structural characterization of AcrIE3 have significant implications for both phage biology and biotechnology:

• Phage Defense: It illustrates a specific evolutionary strategy ("anti-CRISPR") used by phages to overcome bacterial adaptive immunity.
• Gene Editing: As a potent "off-switch" for the Type I-E CRISPR-Cas system, AcrIE3 can be developed into a tool for regulating gene editing activities. This allows for precise temporal control of CRISPR activity, potentially reducing off-target effects in therapeutic applications.

Structural highlights

The crystal structure of AcrIE3 (PDB: 8HEL) reveals key features that enable its inhibitory function:

• Helical Bundle: The protein adopts a compact all-helical fold composed of three alpha-helices and a short 3_10 helix.
• Surface Charge: A striking feature of AcrIE3 is its highly negatively charged surface. This acidic surface mimics the phosphate backbone of DNA.
• Key Residues: Mutational analysis has identified specific acidic residues, such as , , and , as critical for the interaction. These residues interact with the positively charged DNA-binding cleft of the Cas8e subunit, effectively competing with the target DNA for binding.