8HNT

Crystal structure of anti-CRISPR protein AcrIIC4 bound to HpaCas9-sgRNA surveillance complex

Summary for 8HNT

• Entry DOI: 10.2210/pdb8hnt/pdb
• Descriptor: CRISPR-associated endonuclease Cas9, sgRNA, anti-CRISPR protein AcrIIC4 (3 entities in total)
• Functional Keywords: cas9, antimicrobial protein, rec lobe, immune system
• Biological source: Haemophilus parainfluenzae; More
• Total number of polymer chains: 3
• Total formula weight: 172649.29

Authors

Sun, W.,Cheng, Z.,Wang, Y. (deposition date: 2022-12-08, release date: 2023-07-19, Last modification date: 2024-05-29)

Primary citation

Sun, W.,Cheng, Z.,Wang, J.,Yang, J.,Li, X.,Wang, J.,Chen, M.,Yang, X.,Sheng, G.,Lou, J.,Wang, Y.
AcrIIC4 inhibits type II-C Cas9 by preventing R-loop formation.
Proc.Natl.Acad.Sci.USA, 120:e2303675120-e2303675120, 2023

PubMed Abstract: Anti-CRISPR (Acr) proteins are encoded by phages and other mobile genetic elements and inhibit host CRISPR-Cas immunity using versatile strategies. AcrIIC4 is a broad-spectrum Acr that inhibits the type II-C CRISPR-Cas9 system in several species by an unknown mechanism. Here, we determined a series of structures of Cas9 (HpaCas9)-sgRNA in complex with AcrIIC4 and/or target DNA, as well as the crystal structure of AcrIIC4 alone. We found that AcrIIC4 resides in the crevice between the REC1 and REC2 domains of HpaCas9, where its extensive interactions restrict the mobility of the REC2 domain and prevent the unwinding of target double-stranded (ds) DNA at the PAM-distal end. Therefore, the full-length guide RNA:target DNA heteroduplex fails to form in the presence of AcrIIC4, preventing Cas9 nuclease activation. Altogether, our structural and biochemical studies illuminate a unique Acr mechanism that allows DNA binding to the Cas9 effector complex but blocks its cleavage by preventing R-loop formation, a key step supporting DNA cleavage by Cas9.

PubMed: 37494395
DOI: 10.1073/pnas.2303675120

Experimental method

X-RAY DIFFRACTION (3.06 Å)