Structural and mechanistic investigation of the anti-CRISPR protein AcrIE5 using NMR spectroscopy and AlphaFold modeling.

The CRISPR-Cas system employs RNA-guided endonucleases to protect bacteria and archaea from invading bacteriophages and plasmids. In response, bacteriophages have evolved anti-CRISPR proteins that inhibit diverse types of the CRISPR-Cas system. AcrIE5 was discovered from the mobile genetic elements of Pseudomonas aeruginosa, and potently inhibits the type I-E CRISPR-Cas system of P. aeruginosa. Here, we determined the solution structure of AcrIE5 using NMR spectroscopy, which adopts a novel αβ fold comprising three α-helices and two β-strands. AcrIE5 harbors a mobile loop between the β-strands that is conserved among homologs encoded by MGEs infecting Pseudomonas species, but truncated in homologs from MGEs of other bacteria. AlphaFold correctly reproduced the experimental structure of AcrIE5 and predicted its binding at a cleft formed by Cas8e, Cas7e, and Cas5e within the P. aeruginosa type I-E Cascade. The mobile loop and α-helices of AcrIE5 mediated key interactions with Cas8e at the PAM recognition site, as well as with adjacent Cas7e and Cas5e. AcrIE5 did not bind individual subunits of Cascade with high affinity, suggesting that it recognizes a composite interface of the functional Cascade assembly. Taken together, our findings suggest that AcrIE5 may compete with DNA binding to the PAM recognition site of type I-E Cascade, similar to AcrIE3 and AcrIE4, and also highlight a potential functional role of the conserved mobile loop in host-specific anti-CRISPR activity.