AcrF3 (AcrIF3) is a 139-residue homodimeric anti-CRISPR protein of Pseudomonas phage JBD5. It binds the type I-F Cas2/3 helicase-nuclease, stabilizes an ADP-bound state and masks surfaces required for recruitment to the DNA-bound Csy surveillance complex. Target recognition by Csy is retained, but downstream DNA destruction and Cas3-dependent primed spacer acquisition are inhibited. The target-bound, nuclease-free surveillance complex can consequently repress transcription.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0046872 metal ion binding | IEA GO_REF:0000043 | MARK AS OVER ANNOTATED | Summary: The electronic metal-binding annotation is associated with calcium contacts in a crystal structure, but physiological metal dependence has not been established. Reason: Retain MARK_AS_OVER_ANNOTATED: deposited metal contacts are real observations, but they do not establish a physiological metal-binding function. The demonstrated inhibitory mechanism is sequestration of Cas2/3 and masking of recruitment surfaces. A metal requirement is not established by the cited structural and inhibition work. Supporting Evidence: PMID:27585537 our structural analysis of the Cas3-AcrF3 complex revealed that the AcrF3 dimer binds to HD, Linker and CTD domains of the Cas3 protein, therefore blocking the replaced non-complementary DNA access of Cas3 PMID:27455460 AcrF3 forms a homodimer that locks PaCas3 in an ADP-bound form, blocks the entrance of the DNA-binding tunnel in the helicase domain, and masks the linker region and C-terminal domain of PaCas3, thereby preventing recruitment by Cascade and inhibiting the type I-F CRISPR-Cas system. |
| GO:0140311 protein sequestering activity | IDA PMID:27455460 Structural basis of Cas3 inhibition by the bacteriophage pro... | NEW | Summary: The AcrF3 homodimer binds the host Cas2/3 helicase-nuclease and masks the linker region and C-terminal domain that the Csy surveillance complex uses to recruit it, so Cas2/3 can neither interact with its partner complex nor reach the R-loop where it acts. Two independent crystal structures of the AcrF3-Cas3 complex, both solved with this exact accession, establish the interaction and the occluded surfaces. Reason: GOA holds no process or mechanism term for L7P7R7 at all, only the keyword-derived metal ion binding row. GO:0140311 is asserted because its definition fits literally and in both of its clauses, namely binding a protein to prevent it from interacting with other partners, and inhibiting its localization to the area of the cell or complex where it is active. This is the one anti-CRISPR mechanism in modules/anti_crispr_suppression.yaml for which GO already has a precise molecular-function term, so no new term is proposed. The evidence code is IDA rather than ISS because PDB 5B7I, 5GNF and 5GQH all carry chains spanning residues 1 to 139 of this accession, so the structural work was done on this sequence. GO:0005515 protein binding would be the uninformative alternative and is avoided. Supporting Evidence: PMID:27455460 AcrF3 forms a homodimer that locks PaCas3 in an ADP-bound form, blocks the entrance of the DNA-binding tunnel in the helicase domain, and masks the linker region and C-terminal domain of PaCas3, thereby preventing recruitment by Cascade and inhibiting the type I-F CRISPR-Cas system. PMID:27585537 our structural analysis of the Cas3-AcrF3 complex revealed that the AcrF3 dimer binds to HD, Linker and CTD domains of the Cas3 protein, therefore blocking the replaced non-complementary DNA access of Cas3 PMID:26416740 The third anti-CRISPR protein operates by binding to the Cas3 helicase-nuclease and preventing its recruitment to the DNA-bound CRISPR-Cas complex. |
| GO:0098672 symbiont-mediated suppression of host CRISPR-cas system | IDA PMID:26416740 Multiple mechanisms for CRISPR-Cas inhibition by anti-CRISPR... | NEW | Summary: Sequestering Cas2/3 suppresses the host type I-F CRISPR-Cas system at the interference step and, because recruitment of Cas2/3 is also required for primed spacer acquisition, at the memory-updating step as well. GO:0098672 is the correct and maximally specific process term. Reason: AcrF3 directly performs CRISPR suppression by sequestering Cas2/3 and preventing its recruitment to the target-bound Csy complex. The process is established by the inhibition and recruitment assays, independently of the annotation state of other inhibitors. Supporting Evidence: PMID:26416740 The third anti-CRISPR protein operates by binding to the Cas3 helicase-nuclease and preventing its recruitment to the DNA-bound CRISPR-Cas complex. PMID:27585537 DNA degradation via inhibiting recruitment of Cas3 by Csy complex. In addition, inhibition of Cas3-guided DNA degradation also blocks primed spacer acquisition. In summary, the AcrF3 dimer overcomes the CRISPR system at both the crRNA interference and the primed acquisition stages. |
| GO:0030430 host cell cytoplasm | IC PMID:26416740 Multiple mechanisms for CRISPR-Cas inhibition by anti-CRISPR... | NEW | Summary: AcrF3 is translated from the JBD5 genome inside the infected Pseudomonas aeruginosa cell and sequesters the host Cas2/3 protein there. Inhibition, and the conversion of the host system into a transcriptional repressor, are both demonstrated in living cells, so the protein must act in the host cytoplasm; the structures were determined in vitro, so the location is inferred rather than measured. Reason: The UniProt taxon for this entry is the phage, so the host-qualified component term is the correct one and GO:0005737 cytoplasm would be wrong for a viral protein acting in another organism. The same assignment with the same IC code was made for the type I-F inhibitors AcrF8 (genes/BPZF4/AcrF8) and gp30 (genes/BPD31/orf30). Supporting Evidence: PMID:26416740 In vivo, this anti-CRISPR can convert the CRISPR-Cas system into a transcriptional repressor PMID:26416740 The third anti-CRISPR protein operates by binding to the Cas3 helicase-nuclease and preventing its recruitment to the DNA-bound CRISPR-Cas complex. |
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Download this section (compressed HTML)Q: Does AcrF3 sequestration of Cas2/3 leave the host with a functional sequence-specific DNA-binding module that has physiological consequences of its own, beyond the reported transcriptional repression, for instance interference with replication at self-targeting loci?
Suggested experts: Alan R. Davidson, Karen L. Maxwell
Q: Is the Ca(2+) seen at the AcrF3 N-terminus and surface in PDB 5GNF conserved across AcrF3-family members, or is it unique to that crystal form, which would settle the GO:0046872 question definitively?
Suggested experts: Yanli Wang
Q: Cas3 sequestration has no counterpart against class 2 systems, where recognition and cleavage sit in one protein. Is that why class 2 inhibitors converged instead on domain blockade, as AcrIIC1 does, and guide destruction, as AcrVA1 does?
Suggested experts: Joseph Bondy-Denomy
Experiment: Measure AcrF3 dimerisation by size-exclusion chromatography with inline light scattering, and AcrF3-Cas2/3 affinity by isothermal titration calorimetry, in buffers with saturating Ca(2+), with no added divalent cation, and with EDTA, then test inhibition of Cas2/3-dependent plasmid degradation under the same three conditions. Calcium-independence across all three readouts would support removing the keyword outright rather than only marking the annotation over-annotated.
Hypothesis: Calcium is not required for AcrF3 function, so the GO:0046872 annotation reflects crystallisation conditions rather than biology.
Type: metal-dependence titration with paired binding and activity readouts
Experiment: Reconstitute the licensed interference intermediate by assembling Csy complex on a matched protospacer duplex and recruiting purified Cas2/3, then add AcrF3 either before or after recruitment and score target degradation and complex composition by native gel and mass photometry. An order-of-addition effect is the prediction; its absence would mean AcrF3 is an active disassembly factor rather than a sequestering agent, which would change the molecular function assigned here.
Hypothesis: AcrF3 can only intercept free Cas2/3 and cannot displace an already recruited nuclease.
Type: order-of-addition reconstitution assay
Experiment: Challenge Pseudomonas aeruginosa PA14 carrying an intact type I-F system with a partially mismatched protospacer plasmid while inducing either AcrF3 or the Csy-blocking gp30, then deep sequence the CRISPR arrays for newly acquired spacers. The mechanisms predict different outcomes, since a Csy blocker prevents the initial recognition that priming starts from whereas AcrF3 permits recognition but removes the nuclease, so comparing them tests whether priming requires Cas2/3 activity or only target engagement.
Hypothesis: AcrF3 blocks primed spacer acquisition in vivo, and does so to a greater extent than inhibitors that act on the surveillance complex.
Type: CRISPR array deep sequencing with paired inhibitor comparison
What is not known — curated, literature-grounded statements of the open unknowns (the inverse of core functions).
Gap: It is undetermined whether AcrF3 can extract Cas2/3 that is already engaged with a licensed R-loop, or whether it can only intercept free Cas2/3 before recruitment.
OPEN BIOLOGY
What is known: That AcrF3 prevents recruitment, and which Cas3 surfaces it masks, are both established structurally. The ambiguity concerns the order of events, not the interaction.
Significance: The two possibilities differ in what they demand of the phage. Intercepting free Cas2/3 requires only that AcrF3 reach inhibitory concentration before the first target is licensed, which is a race the phage can win by expressing early. Displacing an engaged nuclease would make the inhibitor effective at any point in the infection, and would also explain how a phage rescues itself after a delay.
What would resolve it: Assemble Csy complex on a target duplex, recruit Cas2/3, then add AcrF3 and follow dissociation and DNA degradation, comparing against addition of AcrF3 before recruitment.
Provenance (the field's own admissions):
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