AcrIIA2 of Listeria phage LP-101 is a 123-residue homologue of characterized Listeria prophage anti-CRISPR proteins. Those homologues bind guide-loaded Cas9 and occupy the target-DNA recognition surface through an acidic DNA-mimicking interface, inhibiting type II-A interference. The LP-101 protein differs at nine positions from the assayed J0161 reference protein, so its mechanism and potency are inferred from family membership and close sequence similarity rather than measured directly on this accession.
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 | ISS PMID:28041849 Inhibition of CRISPR-Cas9 with Bacteriophage Proteins. | NEW | Summary: The AcrIIA2 family inhibits both the native Listeria monocytogenes Cas9 and the heterologous Streptococcus pyogenes Cas9, in bacteria and in human cells, and the crystal structure of the inhibited complex has been solved. GO:0098672 is the correct and maximally specific process term. This accession is a 92.7 percent identical orthologue of the sequence on which that work was done, so the claim is transferred rather than demonstrated here. Reason: This is a prior ISS proposal, not direct experimental evidence on the LP-101 protein. The characterized J0161 protein AEO04363.1 is identical at 114 of 123 positions; UniProt family signatures independently support AcrIIA2 membership. Transfer of the inhibitory role is reasonable, but effects of the nine substitutions on potency and orthologue specificity remain untested. Supporting Evidence: PMID:28041849 these data demonstrate the utility of the AcrIIA2 and AcrIIA4 proteins to inhibit the function of an orthologous Cas9 in heterologous hosts PMID:28041849 AcrIIA2- (AEO04363.1), AcrIIA3- (CBY03209.1) and AcrIIA4- (AEO04689.1) homologous protein sequences were acquired by BLASTp searches PMID:28448066 Recently, two anti-CRISPR proteins (AcrIIA2 and AcrIIA4 from Listeria monocytogenes prophages) were identified, both of which inhibit Streptococcus pyogenes Cas9 (SpyCas9) and L. monocytogenes Cas9 activity in bacteria and human cells. |
| GO:0043021 ribonucleoprotein complex binding | ISS PMID:28448066 Structural basis of CRISPR-SpyCas9 inhibition by an anti-CRI... | NEW | Summary: AcrIIA2 binds Cas9 only once the guide RNA is loaded, and the solved structure is of the ternary AcrIIA2-SpyCas9-sgRNA complex, so the entity engaged is a ribonucleoprotein rather than a free protein. Reason: Asserted because it is literally true of the family and checkable against the deposited ternary complex, and because the same term was used for the analogous inhibitors AcrF8 (genes/BPZF4/AcrF8) and AcrIIA4 (genes/LISMN/acrIIA4). It is deliberately not offered as the core activity, since it records what is bound and not the mimicry that makes the binding inhibitory. GO:0005515 protein binding is avoided as uninformative. ISS for the same reason as the process row, with AEO04363.1 as the source sequence. Supporting Evidence: PMID:28448066 Our data show that AcrIIA2 and AcrIIA4 interact with SpyCas9 in a sgRNA-dependent manner. PMID:30606466 We show that AcrIIA2 binds SpyCas9 at a position similar to the target DNA binding region. |
| GO:0030430 host cell cytoplasm | IC PMID:28041849 Inhibition of CRISPR-Cas9 with Bacteriophage Proteins. | NEW | Summary: AcrIIA2 is translated from the phage genome inside the infected Listeria cell and acts on the host Cas9-guide RNA complex, a cytoplasmic ribonucleoprotein. Inhibition of Cas9 is demonstrated inside living cells, so the protein must reach the host cytoplasm; the structure was determined in vitro, so the location is inferred rather than measured. Reason: This UniProt entry is assigned to the phage taxon, Listeria phage LP-101, so the host-qualified component term is the right one and GO:0005737 cytoplasm would be wrong for a viral protein acting in another organism. The contrast with genes/LISMN/acrIIA4 is deliberate; that entry is assigned to Listeria monocytogenes itself because the gene sits in a resident prophage, and takes GO:0005737 instead. IC rather than IDA because no localization experiment exists. Supporting Evidence: PMID:28041849 these data demonstrate the utility of the AcrIIA2 and AcrIIA4 proteins to inhibit the function of an orthologous Cas9 in heterologous hosts PMID:30606466 AcrIIA2 interacts with the protospacer adjacent motif (PAM) recognition residues of Cas9, preventing target double-stranded DNA (dsDNA) detection. |
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Download this section (compressed HTML)Q: AcrIIA2 and AcrIIA4 occlude the same Cas9 surface with different binding motifs and no sequence relationship. Does the Cas9 PAM-interacting domain show signatures of selection driven by both, or has it been unable to escape because the mimicked feature, the phosphate backbone, cannot be abandoned?
Suggested experts: Yanli Wang, Joseph Bondy-Denomy
Q: Do the nine substitutions separating the LP-101 orthologue from the assayed prophage protein alter potency against Lmo Cas9, and is the Listeria phage-versus-prophage split reflected in two functionally distinct AcrIIA2 subfamilies?
Suggested experts: Yanli Wang
Q: Why does the apparent effect of AcrIIA2 on Cas9 cleavage exceed its effect on DNA binding, if the mechanism is occlusion of the PAM-recognition site upstream of both?
Suggested experts: Joseph Bondy-Denomy
Experiment: Express LP101_024 and AEO04363.1 side by side, then measure Cas9 inhibition on three readouts at matched protein concentrations, namely isothermal titration calorimetry against sgRNA-loaded SpyCas9, in vitro plasmid cleavage, and plasmid interference rescue in a Cas9-competent host. Equivalence would retrospectively justify the ISS annotations here and let them be upgraded; divergence would mean the module's exemplar must be swapped for the assayed sequence.
Hypothesis: The LP-101 orthologue is functionally equivalent to the assayed Listeria prophage AcrIIA2, so the nine substitutions are neutral with respect to Cas9 inhibition.
Type: side-by-side orthologue potency comparison
Experiment: Titrate AcrIIA2 against sgRNA-loaded dCas9 across a range of target duplex concentrations and fit the inhibition surface to competitive and non-competitive models. A mimic should lose apparent potency as duplex concentration rises; a failure of that prediction would mean the acidic surface binds at the same place as DNA without being outcompeted by it, which is a different mechanism and would need different wording in the proposed molecular function.
Hypothesis: AcrIIA2 inhibition is competitive with target DNA, which is the operational test of the mimicry claim.
Type: competitive binding isotherm analysis
Experiment: Delete LP101_024 from phage LP-101 and compare plaquing efficiency on a Listeria monocytogenes host with an intact type II-A system against an isogenic cas9 deletion, with complementation in trans as the control. This is the necessity-and-sufficiency test that grounded the original anti-CRISPR assignments, and it has not been done for a free Listeria phage; it would also convert these ISS annotations into experimental ones on this very accession.
Hypothesis: acrIIA2 is required for LP-101 to replicate on a Cas9-competent Listeria host.
Type: phage gene deletion and plaquing assay
What is not known — curated, literature-grounded statements of the open unknowns (the inverse of core functions).
Gap: No function has been measured on this accession. Every functional claim for A0A059T5F6 is transferred from the 92.7 percent identical Listeria monocytogenes prophage protein AEO04363.1, and GOA holds no annotations for it at all.
OPEN CURATIONBIOLOGY
What is known: The AcrIIA2 family's mechanism is firmly established by a crystal structure of the inhibited complex and by in vivo inhibition of two Cas9 orthologues. What is unmeasured is whether this particular phage orthologue, with its nine substitutions, retains full potency, and whether the LP-101 phage actually depends on it.
Significance: This accession is the gene-product-level exemplar for the AcrIIA2 convergence case in modules/anti_crispr_suppression.yaml, and the module cites the structural paper as evidence for it. A reader following the accession reaches an unreviewed entry whose protein name is a ProtNLM machine prediction, with no experiment on the sequence itself. This is the same accession-level slippage the module already records as an open gap for the AcrIII-1 exemplar.
What would resolve it: Either assay the LP-101 protein directly, by expressing LP101_024 and testing inhibition of Lmo Cas9 and SpyCas9 and deleting it from the phage, or replace the module's gene-product selector with the assayed prophage sequence and keep LP101_024 as a family member.
Provenance (the field's own admissions):
Gap: Whether AcrIIA2 inhibits only target licensing, as pure PAM-site occlusion predicts, or additionally interferes with the cleavage step, is undetermined.
OPEN BIOLOGY
What is known: The structure establishes occupancy of the PAM-recognition residues and competition with the target duplex. The ambiguity is about a possible second, post-licensing effect.
Significance: Pure occlusion of the PAM-reading surface should affect DNA binding and DNA cleavage equally, because nothing downstream can happen without licensing. The genetics showed a larger effect on cleavage than on dCas9 binding, which if real would mean AcrIIA2 belongs partly in the module's nuclease-neutralisation part as well as in its recognition-blockade part, and would move it off the pure AcrIIA4 analogy.
What would resolve it: Compare AcrIIA2 inhibition of dCas9 target binding and of wild-type Cas9 cleavage side by side with purified components and matched stoichiometries, rather than in a heterologous reporter system.
Provenance (the field's own admissions):
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