Structural insights into promoter recognition by Aca7

CRISPR-Cas systems provide adaptive immunity to bacteria, although bacteriophages counter these defenses with anti-CRISPR (Acr) proteins. Acr expression is frequently regulated by anti-CRISPR associated (Aca) proteins, which repress transcription by binding inverted repeat (IR) sequences in operon promoters. Here, we report the first identification of an IR motif within the AcrIF11-Aca7 operon promoter from Halomonas caseinilytica and present the crystal structure of Aca7 bound to this IR DNA. Biochemical assays demonstrated that Aca7 specifically recognizes the IR element, and structural analysis revealed a symmetric Aca7 dimer engaging both major grooves via helix-turn-helix motifs while stabilizing DNA bending through minor groove contacts. Residue-level interactions, including those mediated by R38, Q42, K46, and K49, establish a detailed basis for sequence-specific recognition. Comparison with Aca2 highlights distinct dimer architectures and DNA deformation strategies among Aca proteins. Our findings uncover the molecular mechanism by which Aca7 represses AcrIF11 expression and broaden the understanding of Aca-mediated transcriptional regulation.