AcrIIA4 is an 87-residue prophage anti-CRISPR protein recorded in Listeria monocytogenes. Its acidic surface mimics DNA and binds guide-loaded Cas9 at the PAM-recognition region, with a loop also occluding the non-target-strand path into the RuvC pocket. This prevents new target-DNA engagement. In tested SpyCas9 assays it binds at 1:1 stoichiometry and does not displace DNA from a preformed Cas9-guide-DNA complex, so inhibitory efficacy depends on timing and target occupancy. Activity against other Cas9 orthologues is limited to those experimentally shown to be susceptible.
Definition: A molecular function inhibitor activity in which a protein presents a surface whose shape and charge distribution imitate a nucleic acid and occupies its binding site on a target protein or protein-containing complex, thereby preventing engagement of the genuine nucleic acid ligand.
Justification: The proposed term distinguishes inhibition through nucleic acid mimicry from other forms of inhibition. Existing nuclease inhibitor or molecular function inhibitor terms can describe the broader activity, and ribonucleoprotein complex binding describes the interaction. The proposal adds the mechanism shared by structurally unrelated AcrIF2, AcrIIA2 and AcrIIA4 proteins. It does not imply rapid reversibility, displacement of pre-bound substrate, or a universal requirement for inhibitor molar excess; occupancy depends on affinity, assembly state and order of addition.
Parent term: molecular function inhibitor activity
Supporting Evidence:
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0098672 symbiont-mediated suppression of host CRISPR-cas system | IDA PMID:28041849 Inhibition of CRISPR-Cas9 with Bacteriophage Proteins. | ACCEPT | Summary: AcrIIA4 was identified as one of four prophage-encoded type II-A Cas9 inhibitors in Listeria monocytogenes and shown to block both the native Lmo Cas9 and the heterologous Streptococcus pyogenes Cas9 in bacteria and in human cells. GO:0098672 is the correct and maximally specific process term for this activity, and the accession is the exact sequence used in the work (AEO04689.1, identical over all 87 residues), so the IDA code is properly grounded. Reason: The original experimental annotation is supported by the native inhibitor discovery and subsequent structural and biochemical studies. The reference sequence AEO04689.1 matches this accession over all 87 residues. Supporting Evidence: PMID:28041849 This analysis led to the discovery of four unique type II-A CRISPR-Cas9 inhibitor proteins encoded by Listeria monocytogenes prophages. PMID:28041849 Two of these inhibitors also blocked the widely used Streptococcus pyogenes Cas9 when assayed in Escherichia coli and human cells. |
| GO:0043021 ribonucleoprotein complex binding | IDA PMID:28448066 Structural basis of CRISPR-SpyCas9 inhibition by an anti-CRI... | NEW | Summary: AcrIIA4 binds Cas9 only after the guide RNA is loaded, because the composite surface it docks onto is created by the conformational change that sgRNA binding induces. The crystal structure is of the ternary AcrIIA4-SpyCas9-sgRNA complex, so the entity bound is a ribonucleoprotein, not a free protein. Reason: Guide dependence is a defining feature of this inhibitor and is reported independently by two structural groups, but GOA holds no molecular function term for A0A247D711 at all. GO:0043021 is asserted because it is literally true and checkable, and because the same term was used for the analogous type I-F inhibitor AcrF8 in genes/BPZF4/AcrF8. It is deliberately not offered as the core activity, since it records what is bound and not the mimicry that makes the binding inhibitory; the core activity is carried by the proposed term instead. GO:0005515 protein binding is avoided as uninformative. Supporting Evidence: PMID:28448066 Our data show that AcrIIA2 and AcrIIA4 interact with SpyCas9 in a sgRNA-dependent manner. PMID:28448066 Structural comparison reveals that formation of the AcrIIA4-binding site of SpyCas9 is induced by sgRNA binding. PMID:28602637 Selective Binding of AcrIIA4 to sgRNA-bound SpyCas9 |
| GO:0005737 cytoplasm | IC PMID:28041849 Inhibition of CRISPR-Cas9 with Bacteriophage Proteins. | NEW | Summary: AcrIIA4 is translated from a prophage integrated in the Listeria monocytogenes chromosome and acts on the host's own Cas9-guide RNA complex, which is a cytoplasmic ribonucleoprotein. The location is inferred by curator from the binding partner and from the fact that inhibition is demonstrated inside living cells; both published structures were determined in vitro, so there is no direct localization experiment. Reason: GO:0030430 host cell cytoplasm, which was used for the phage-taxon inhibitor AcrF8, is not appropriate here. This UniProt entry is assigned to Listeria monocytogenes itself, not to a phage, because the gene lies in a resident prophage; the cell whose cytoplasm the protein occupies is therefore its own cell under that taxon assignment, not a separate host. GO:0005737 is the correct component term for that situation. Evidence is IC rather than IDA because localization was inferred, not measured. Supporting Evidence: PMID:28041849 This analysis led to the discovery of four unique type II-A CRISPR-Cas9 inhibitor proteins encoded by Listeria monocytogenes prophages. PMID:28041849 Two of these inhibitors also blocked the widely used Streptococcus pyogenes Cas9 when assayed in Escherichia coli and human cells. |
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Download this section (compressed HTML)Q: How do native AcrIIA4 expression timing and Cas9 target occupancy determine protection during prophage induction?
Experiment: Measure AcrIIA4 and Cas9 abundance alongside guide loading and target occupancy through a natural Listeria prophage induction time course.
What is not known — curated, literature-grounded statements of the open unknowns (the inverse of core functions).
Gap: The concentration and timing of AcrIIA4 production relative to free and DNA-bound Cas9 during native lysogeny and induction remain incompletely quantified.
OPEN BIOLOGY
What is known: In vitro order-of-addition experiments show inhibition before target binding and no displacement of preformed Cas9-guide-DNA complexes. Native abundance measurements are a separate issue.
Significance: The fraction of accessible, guide-loaded Cas9 depends on infection timing as well as affinity and protein abundance. A universal inhibitor molar-excess requirement does not follow from 1:1 binding.
What would resolve it: Quantify AcrIIA4 and Cas9 abundance and occupancy through native prophage induction.
Provenance (the field's own admissions):
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