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Mode of Action (MoA)
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binds to the Cas7.4/7.6f subunits of the Csy complex, inducing strong non-specific DNA binding activity.
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Evidence
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AcrIF14, encoded in phage Mcat5, was validated through ectopic expression in type I-F CRISPR-active strains (ECO_0000017). It restored DMS3m replication, confirming inhibition. Cryo-EM structures of the Csy–AcrIF14 complex (ECO_0006181) showed two AcrIF14 molecules engaging the Cas7.4f and Cas7.6f subunits, sterically blocking crRNA–DNA hybridization. Key interface residues (e.g., R84, Y89, E91, F104, Y105) were identified via mutational analysis; pull-down (ECO_0006249) assays and cleavage assays confirmed their importance for inhibition. Additionally, AcrIF14 induced strong non-sequence-specific dsDNA binding when complexed with Csy, evidenced by electrophoretic mobility shift assays (EMSA, ECO_0000096) using dsDNA (non-specific). This DNA-binding activity was abolished in AcrIF14 mutants targeting its positively charged N-terminal patches, and in Csy complexes bearing mutations in the Cas8f PAM-recognition loop (K247E, N250D) or the R-loop Binding Channel (R207E/R219E/R258E).
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MoA Category
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binds and inhibits host defence system
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Subtype(s) of the defence system(s) inhibited by the protein
Defence Subtype
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Pseudomonas aeruginosa type I-F CRISPR-Cas
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Relevant publication(s)
DOI
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10.1126/science.aau5174
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Other components of the anti-defence system
Multicomponent System
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-
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Known structure in PDB
PDB ID
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7DU0
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Genome(s) encoding the protein
Protein Source
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Moraxella phage Mcat5
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Defence system(s) inhibited by the protein
Defences
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CRISPR-Cas
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