Biochemical characterization of type I-E anti-CRISPR proteins, AcrIE2 and AcrIE4

Abstract

In bacteria and archaea, CRISPRs and Cas proteins constitute an adaptive immune system against invading foreign genetic materials, such as bacteriophages and plasmids. To counteract CRISPR-mediated immunity, bacteriophages encode anti-CRISPR (Acr) proteins that neutralize the host CRISPR–Cas systems. Several Acr proteins that act against type I-E CRISPR–Cas systems have been identified. Here, we describe the biochemical characterization of two type I-E Acr proteins, AcrIE2 and AcrIE4. We determined the crystal structure of AcrIE2 using single-wavelength anomalous diffraction and performed a structural comparison with the previously reported AcrIE2 structures solved by different techniques. Binding assays with type I-E Cas proteins were carried out for the target identification of AcrIE2. We also analyzed the interaction between AcrIE4 and its target Cas component using biochemical methods. Our findings corroborate and expand the knowledge on type I-E Acr proteins, illuminating diverse molecular mechanisms of inhibiting CRISPR-mediated prokaryotic anti-phage defense.