Molecular basis of transcriptional repression of anti-CRISPR by anti-CRISPR-associated 2

Abstract

CRISPR–Cas systems are well known host defense mechanisms that are conserved in bacteria and archaea. To counteract CRISPR–Cas systems, phages and viruses have evolved to possess multiple anti-CRISPR (Acr) proteins that can inhibit the host CRISPR–Cas system via different strategies. The expression of acr genes is controlled by anti-CRISPR-associated (Aca) proteins that bind to an upstream promoter and regulate the expression of acr genes during transcription. Although the role of Aca as a transcriptional repressor has been demonstrated, the mechanism of action of Aca has not been determined. Here, the molecular mechanism underlying the Aca2-mediated transcriptional control of acr genes was elucidated by determining the crystal structure of Aca2 from Oceanimonas smirnovii at a high resolution of 1.92 Å. Aca2 forms a dimer in solution, and dimerization of Aca2 is critical for specific promoter binding. The promoter-binding strategy of dimeric Aca2 was also revealed by performing mutagenesis studies. The atomic structure of the Aca family shown in this study provides insights into the fine regulation of host defense and immune-escape mechanisms and also demonstrates the conserved working mechanism of the Aca family.

Molecular basis of dual anti-CRISPR and auto-regulatory functions of AcrIF24

“…Although various Acrs inhibit CRISPR-Cas activity in monomeric form, previous studies have shown that the dimeric form of Acrs is often critical for their activity ( 29 , 53 , 54 ). Similarly, some Aca proteins have been shown to function as dimers ( 31 , 33 , 55 ). Given the possible dimeric form of AcrIF24 as judged by our SEC experiment, we used multi-angle light scattering (MALS) to confirm the stoichiometry by determining the absolute molecular mass of AcrIF24 in solution.…”

“…Many DNA binding proteins with HTH motifs function as dimers. Indeed, dimerization of Aca proteins is critical for the recognition of inverted repeat (IR) DNA sequences in the promoter region of acr operons ( 52 , 55 , 58 ). In agreement, AcrIF24 bound to a short IR sequence in the promoter region of the acr operon and repressed transcription.…”