{"gene":"ZYG11B","run_date":"2026-06-11T09:02:07","timeline":{"discoveries":[{"year":2021,"finding":"Crystal structures of ZYG11B bound to various Gly/N-degrons reveal that ZYG11B uses its armadillo (ARM) repeats to form a deep and narrow cavity that engages mainly the first four residues of Gly/N-degrons, with the α-amino group of the degron accommodated in an acidic pocket via five conserved hydrogen bonds, establishing the structural basis for specific Gly/N-degron recognition by the CRL2ZYG11B E3 ubiquitin ligase complex.","method":"X-ray crystallography of ZYG11B bound to Gly/N-degron peptides, combined with biochemical binding assays","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structures with multiple ligands plus biochemical validation; single lab but multiple orthogonal methods","pmids":["34214466"],"is_preprint":false},{"year":2021,"finding":"SARS-CoV-2 ORF10 physically interacts with ZYG11B (the substrate receptor of CRL2ZYG11B), and the N-terminus of ORF10 is critical for this interaction; however, ORF10 does not function to inhibit or hijack CRL2ZYG11B, and ZYG11B (and its paralog ZER1) are dispensable for SARS-CoV-2 infection in cultured cells.","method":"Co-immunoprecipitation, N-terminal deletion analysis, ZYG11B/ZER1 knockout cell infection assays","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP plus KO functional assay in a single lab, two orthogonal methods","pmids":["33827988"],"is_preprint":false},{"year":2020,"finding":"A truncating mutation in ZYG11B (p.Glu537*) produces a protein with altered subcellular localization in HeLa cells compared to wild-type ZYG11B; knockdown of the zebrafish ZYG11B homologue disrupts craniofacial cartilage architecture and notochord development, and ZYG11B expression regulates the cartilage master regulator SOX6 and is regulated by retinoic acid.","method":"Overexpression and immunofluorescence of wild-type vs. mutant ZYG11B in HeLa cells; morpholino-based knockdown in zebrafish embryos with phenotypic readout; RT-qPCR for SOX6","journal":"Molecular genetics & genomic medicine","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — direct localization experiment with functional consequence plus zebrafish KD phenotype, single lab, two orthogonal model systems","pmids":["32738032"],"is_preprint":false},{"year":2022,"finding":"ZYG11B (as part of CRL2ZYG11B) recognizes not only N-terminal glycine but also small Nt-residues (Ser, Ala, Cys) in vitro; however, Nt-acetylation of Ser, Ala, and Cys by N-terminal acetyltransferases (NATs) shields these residues from ZYG11B recognition in cells, while in NAT-deficient cells ZYG11B/ZER1 targets these unacetylated small Nt-residues for proteasomal degradation. Crystal structures of ZYG11B bound to these small Nt-residues reveal the molecular mechanism of non-acetylated substrate recognition.","method":"In vitro binding assays, cellular degradation assays in NAT-deficient cells, X-ray crystallography","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structures plus in vitro binding plus cellular functional assays, single lab with multiple orthogonal methods","pmids":["36496439"],"is_preprint":false},{"year":2023,"finding":"ZYG11B acts as an amplifier of cGAS-mediated innate immune signaling by enhancing cGAS-DNA binding affinity, potentiating cGAS-DNA condensation, and stabilizing the cGAS-DNA condensed complex, thereby promoting cGAMP production and downstream interferon/cytokine transcription; knockdown of ZYG11B impairs these responses. Additionally, HSV-1 infection induces ZYG11B degradation in a cGAS-independent manner.","method":"ZYG11B knockdown with cGAMP measurement, co-immunoprecipitation/pulldown for cGAS-DNA-ZYG11B interaction, condensation assays, HSV-1 infection assays","journal":"Cell reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — KD with defined biochemical readout plus binding/condensation assays, single lab, multiple orthogonal methods","pmids":["36933219"],"is_preprint":false},{"year":2022,"finding":"Crystal structure of ZYG11B bound to the N-terminal peptide of SARS-CoV-2 ORF10 reveals the structural basis for recognition of the ORF10 N-terminus by ZYG11B's substrate-binding cavity.","method":"X-ray crystallography of ZYG11B–ORF10 N-terminal peptide complex","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — crystal structure from single lab, single method, no accompanying mutagenesis or functional validation reported in abstract","pmids":["35636250"],"is_preprint":false},{"year":2025,"finding":"ZYG11B, as part of the CRL2ZYG11B complex, targets Enterovirus 71 (EV71) structural protein VP1 for proteasomal degradation via K33-linked ubiquitination; mass spectrometry and immunoprecipitation confirmed the ZYG11B–VP1 interaction and identified key domains on ZYG11B required for VP1 binding and CUL2 recruitment. ZYG11B also restricts related enteroviruses (CA6, CA16, EVD68) whose VP1 ubiquitination sites are highly conserved.","method":"Mass spectrometry, co-immunoprecipitation, domain mapping, proteasome inhibitor assays, K33 ubiquitination linkage analysis, viral replication functional assays","journal":"Journal of virology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — MS plus Co-IP plus functional viral assays in single lab, multiple orthogonal methods","pmids":["40135890"],"is_preprint":false},{"year":2026,"finding":"Cryo-EM structures of full-length human ZYG11B in complex with EloB-EloC adaptor and a Gly/N-degron peptide reveal a seahorse-like architecture with distinct interfaces for adaptor binding and substrate engagement. ZYG11B adopts both monomeric and dimeric assemblies, with the dimer stabilizing two substrate-binding sites in opposite orientations. Functional assays demonstrate that interfaces mediating adaptor recruitment, substrate binding, and dimerization are all essential for substrate degradation.","method":"Cryo-EM structure determination of full-length ZYG11B–EloB–EloC–substrate complex; mutagenesis of assembly interfaces with substrate degradation functional assays","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1 / Strong — cryo-EM structures plus interface mutagenesis plus functional degradation assays, single lab with multiple orthogonal methods","pmids":["41917018"],"is_preprint":false},{"year":2024,"finding":"Cryo-EM structures of the full CRL2-ZYG11B holoenzyme alone and in complex with a Gly/N-degron peptide from NLRP1 reveal that ZYG11B folds into a Leucine-Rich Repeat domain followed by two armadillo repeat domains that mediate CRL2 assembly and NLRP1 Gly/N-degron recognition. ZYG11B promotes NLRP1 inflammasome activation by recognizing and ubiquitinating the Gly/N-degron exposed after viral protease cleavage of NLRP1; blocking ZYG11B recognition of this degron inhibits viral protease-mediated NLRP1 inflammasome activation.","method":"Cryo-EM structure of CRL2-ZYG11B holoenzyme ± Gly/N-degron peptide; in vitro ubiquitination assays; functional inflammasome activation assays with blocking experiments","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — cryo-EM plus functional assays, but preprint with no peer review; multiple orthogonal methods in single lab","pmids":["bio_10.1101_2024.06.24.600508"],"is_preprint":true},{"year":2025,"finding":"A non-inhibitory DNA aptamer targeting ZYG11B was identified and validated as an E3 ligase warhead for PROTAC-based targeted protein degradation (ZATAC platform), demonstrating that ZYG11B can be recruited to degrade diverse neo-substrates (nucleolin, SOX2, mutant p53-R175H) when bridged via an aptamer, and that cancer-cell-targeted 3WJ-ZATACs achieve dual-target degradation and suppress tumor growth in vivo without noticeable toxicity.","method":"Aptamer selection and binding validation; PROTAC/ZATAC functional degradation assays for multiple target proteins; in vivo tumor xenograft experiments","journal":"Nature communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — functional degradation assays with multiple substrates plus in vivo data, single lab, multiple orthogonal methods","pmids":["40082426"],"is_preprint":false}],"current_model":"ZYG11B is the substrate receptor of the CRL2ZYG11B E3 ubiquitin ligase complex, which uses its armadillo (and leucine-rich) repeat domains to recognize small N-terminal residues—primarily N-terminal glycine (Gly/N-degrons) but also unacetylated Ala, Ser, and Cys—via a conserved acidic pocket, targeting substrates (including NLRP1, viral VP1 proteins, and myristoylation-pathway substrates) for K33/K48-linked ubiquitination and proteasomal degradation; additionally, ZYG11B acts outside its E3 role as a positive regulator of cGAS innate immune signaling by enhancing cGAS-DNA binding and condensation, while the full CRL2ZYG11B holoenzyme assembly involves EloB/EloC adaptors and ZYG11B can form functionally relevant dimers that present two substrate-binding sites."},"narrative":{"mechanistic_narrative":"ZYG11B is the substrate-recognition subunit of the CRL2ZYG11B E3 ubiquitin ligase, which targets proteins bearing small N-terminal residues for proteasomal degradation through the N-degron pathway [PMID:34214466, PMID:bio_10.1101_2024.06.24.600508]. Its armadillo repeats form a deep, narrow cavity whose acidic pocket coordinates the α-amino group of an N-terminal glycine through five conserved hydrogen bonds, accounting for selective Gly/N-degron recognition; the same site also engages unacetylated Ser, Ala, and Cys, residues that N-terminal acetyltransferases normally shield from ZYG11B in cells [PMID:34214466, PMID:36496439]. Full-length ZYG11B folds into a leucine-rich repeat domain followed by two armadillo domains and assembles into the holoenzyme via EloB/EloC adaptors and CUL2, with adaptor-binding, substrate-binding, and dimerization interfaces all required for substrate degradation; ZYG11B can dimerize to present two substrate sites [PMID:41917018, PMID:bio_10.1101_2024.06.24.600508]. Through this activity it ubiquitinates physiological and pathogen-derived substrates: it promotes NLRP1 inflammasome activation by recognizing the Gly/N-degron exposed after viral protease cleavage of NLRP1 [PMID:bio_10.1101_2024.06.24.600508], and it restricts enteroviruses by K33-linked ubiquitination of the structural protein VP1 [PMID:40135890]. Independently of its ligase role, ZYG11B amplifies cGAS-mediated innate immune signaling by enhancing cGAS–DNA binding and condensation to potentiate cGAMP production [PMID:36933219]. ZYG11B has also been repurposed as an E3 warhead for aptamer-bridged targeted protein degradation of neo-substrates [PMID:40082426].","teleology":[{"year":2020,"claim":"Before its biochemical role was defined, ZYG11B was linked to vertebrate development, establishing a phenotypic consequence of disrupting the gene.","evidence":"Mutant-vs-wild-type localization in HeLa cells plus morpholino knockdown in zebrafish with SOX6 expression readout","pmids":["32738032"],"confidence":"Medium","gaps":["Does not connect the developmental phenotype to E3 ligase activity or specific substrates","Morpholino knockdown lacks genetic rescue","Truncating-mutation localization effect not mechanistically explained"]},{"year":2021,"claim":"Crystal structures defined how ZYG11B reads an N-terminal glycine, establishing the structural basis of Gly/N-degron recognition by CRL2ZYG11B.","evidence":"X-ray crystallography of ZYG11B–Gly/N-degron peptide complexes with biochemical binding assays","pmids":["34214466"],"confidence":"High","gaps":["Used isolated armadillo-domain structures rather than the full holoenzyme","Did not address physiological substrate identity","Catalytic ubiquitin transfer not visualized"]},{"year":2021,"claim":"A test of whether SARS-CoV-2 ORF10 hijacks CRL2ZYG11B showed physical interaction via the ORF10 N-terminus but no functional requirement, ruling out a ubiquitin-ligase-hijacking model.","evidence":"Reciprocal Co-IP, N-terminal deletion analysis, and ZYG11B/ZER1 knockout infection assays","pmids":["33827988"],"confidence":"Medium","gaps":["Does not explain why ORF10 binds ZYG11B if not to modulate it","Negative infection result confined to cultured cells"]},{"year":2022,"claim":"Extending the degron repertoire, ZYG11B was shown to recognize unacetylated Ser/Ala/Cys N-termini, integrating N-terminal acetylation status into substrate selection.","evidence":"In vitro binding, degradation assays in NAT-deficient cells, and X-ray crystallography of small-Nt-residue complexes","pmids":["36496439"],"confidence":"High","gaps":["Endogenous substrates exposed in NAT-deficient states not enumerated","Relative in vivo contribution versus paralog ZER1 not resolved"]},{"year":2022,"claim":"A co-crystal structure detailed how ZYG11B's substrate cavity engages the ORF10 N-terminal peptide, structurally rationalizing the earlier interaction.","evidence":"X-ray crystallography of the ZYG11B–ORF10 N-terminal peptide complex","pmids":["35636250"],"confidence":"Medium","gaps":["No accompanying mutagenesis or functional validation","Does not reconcile binding with the lack of functional consequence for infection"]},{"year":2023,"claim":"ZYG11B was found to act outside its E3 role as a positive regulator of cGAS, revealing a moonlighting function in innate immune signaling.","evidence":"ZYG11B knockdown with cGAMP measurement, cGAS-DNA-ZYG11B pulldown, condensation assays, and HSV-1 infection assays","pmids":["36933219"],"confidence":"Medium","gaps":["Mechanism linking ZYG11B binding to condensate stabilization not structurally defined","Whether ligase activity contributes to cGAS regulation unresolved","HSV-1-induced ZYG11B degradation pathway unidentified"]},{"year":2024,"claim":"Holoenzyme cryo-EM with an NLRP1 degron tied ZYG11B's degron recognition to inflammasome biology, showing it ubiquitinates the Gly/N-degron exposed by viral protease cleavage of NLRP1.","evidence":"Cryo-EM of CRL2-ZYG11B ± NLRP1 Gly/N-degron peptide, in vitro ubiquitination, and inflammasome activation/blocking assays (preprint)","pmids":["bio_10.1101_2024.06.24.600508"],"confidence":"Medium","gaps":["Preprint without peer review","Physiological breadth of NLRP1 regulation in primary immune cells not established"]},{"year":2025,"claim":"ZYG11B was shown to restrict enteroviruses by K33-linked ubiquitination of VP1, demonstrating antiviral substrate targeting and mapping the required ZYG11B domains.","evidence":"Mass spectrometry, Co-IP, domain mapping, proteasome inhibitor and K33-linkage analysis, and viral replication assays","pmids":["40135890"],"confidence":"Medium","gaps":["In vivo antiviral relevance not tested","Why K33 rather than K48 linkage is used mechanistically unclear"]},{"year":2025,"claim":"A non-inhibitory aptamer warhead established ZYG11B as a recruitable E3 for targeted protein degradation, enabling neo-substrate destruction in vivo.","evidence":"Aptamer selection, ZATAC degradation assays for nucleolin/SOX2/p53-R175H, and tumor xenograft experiments","pmids":["40082426"],"confidence":"Medium","gaps":["Endogenous-substrate physiology not addressed by this engineering study","Long-term specificity and toxicity beyond xenografts unknown"]},{"year":2026,"claim":"Full-length cryo-EM resolved how ZYG11B assembles with EloB/EloC and dimerizes, establishing the complete architecture required for substrate degradation.","evidence":"Cryo-EM of full-length ZYG11B–EloB–EloC–substrate complex with interface mutagenesis and degradation assays","pmids":["41917018"],"confidence":"High","gaps":["Functional advantage of dimer over monomer in cells not quantified","Dynamics of substrate hand-off to the catalytic module not captured"]},{"year":null,"claim":"How ZYG11B's ligase-dependent N-degron functions and its ligase-independent cGAS regulation are coordinated, and the full endogenous substrate set, remain open.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No comprehensive endogenous substrate catalog","Relationship between developmental phenotypes and defined substrates unestablished","Division of labor with paralog ZER1 unresolved"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[6,8]},{"term_id":"GO:0016874","term_label":"ligase activity","supporting_discovery_ids":[6,8]},{"term_id":"GO:0060089","term_label":"molecular transducer activity","supporting_discovery_ids":[0,3]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[4]}],"localization":[],"pathway":[{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[0,3,7]},{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[4,6,8]}],"complexes":["CRL2ZYG11B"],"partners":["CUL2","ELOB","ELOC","CGAS","NLRP1"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q9C0D3","full_name":"Protein zyg-11 homolog B","aliases":[],"length_aa":744,"mass_kda":83.9,"function":"Serves as substrate adapter subunit in the E3 ubiquitin ligase complex ZYG11B-CUL2-Elongin BC. Acts to target substrates bearing N-terminal degrons for proteasomal degradation with the first four residues of substrates being the key recognition elements (PubMed:33093214, PubMed:34214466, PubMed:35636250). Prefers Nt-Gly but also has the capacity to recognize Nt-Ser, -Ala and -Cys (PubMed:36496439). Involved in the clearance of proteolytic fragments generated by caspase cleavage during apoptosis since N-terminal glycine degrons are strongly enriched at caspase cleavage sites. Also important in the quality control of protein N-myristoylation in which N-terminal glycine degrons are conditionally exposed after a failure of N-myristoylation (PubMed:31273098). In addition, plays a role in the amplification of cGAS to enhance innate immune response. Mechanistically, strengthens the processes of cGAS binding with dsDNA and assembling oligomers and also accelerates and stabilizes cGAS-DNA condensation, thereby enhancing production of antiviral IFNs and inflammatory cytokines (PubMed:36933219)","subcellular_location":"Cytoplasm","url":"https://www.uniprot.org/uniprotkb/Q9C0D3/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/ZYG11B","classification":"Not Classified","n_dependent_lines":27,"n_total_lines":1208,"dependency_fraction":0.022350993377483443},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/ZYG11B","total_profiled":1310},"omim":[{"mim_id":"618675","title":"ZYG11 FAMILY, MEMBER A, CELL CYCLE REGULATOR; ZYG11A","url":"https://www.omim.org/entry/618675"},{"mim_id":"618673","title":"ZYG11 FAMILY, MEMBER B, CELL CYCLE REGULATOR; ZYG11B","url":"https://www.omim.org/entry/618673"},{"mim_id":"617764","title":"ZYG11-RELATED CELL CYCLE REGULATOR; ZER1","url":"https://www.omim.org/entry/617764"},{"mim_id":"606636","title":"NLR FAMILY, PYRIN DOMAIN-CONTAINING 1; NLRP1","url":"https://www.omim.org/entry/606636"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Endoplasmic reticulum","reliability":"Approved"},{"location":"Vesicles","reliability":"Approved"},{"location":"Golgi apparatus","reliability":"Additional"}],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in all","driving_tissues":[{"tissue":"skeletal muscle","ntpm":73.8},{"tissue":"tongue","ntpm":57.8}],"url":"https://www.proteinatlas.org/search/ZYG11B"},"hgnc":{"alias_symbol":["FLJ13456"],"prev_symbol":["ZYG11"]},"alphafold":{"accession":"Q9C0D3","domains":[{"cath_id":"-","chopping":"335-443","consensus_level":"medium","plddt":95.0582,"start":335,"end":443}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9C0D3","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q9C0D3-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q9C0D3-F1-predicted_aligned_error_v6.png","plddt_mean":92.31},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=ZYG11B","jax_strain_url":"https://www.jax.org/strain/search?query=ZYG11B"},"sequence":{"accession":"Q9C0D3","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9C0D3.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9C0D3/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9C0D3"}},"corpus_meta":[{"pmid":"34214466","id":"PMC_34214466","title":"Molecular basis for recognition of Gly/N-degrons by CRL2ZYG11B and CRL2ZER1.","date":"2021","source":"Molecular cell","url":"https://pubmed.ncbi.nlm.nih.gov/34214466","citation_count":36,"is_preprint":false},{"pmid":"33827988","id":"PMC_33827988","title":"ORF10-Cullin-2-ZYG11B complex is not required for SARS-CoV-2 infection.","date":"2021","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/33827988","citation_count":31,"is_preprint":false},{"pmid":"32738032","id":"PMC_32738032","title":"Functional and genetic analyses of ZYG11B provide evidences for its involvement in OAVS.","date":"2020","source":"Molecular genetics & genomic medicine","url":"https://pubmed.ncbi.nlm.nih.gov/32738032","citation_count":28,"is_preprint":false},{"pmid":"36496439","id":"PMC_36496439","title":"CRL2ZER1/ZYG11B recognizes small N-terminal residues for degradation.","date":"2022","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/36496439","citation_count":25,"is_preprint":false},{"pmid":"36933219","id":"PMC_36933219","title":"ZYG11B potentiates the antiviral innate immune response by enhancing cGAS-DNA binding and condensation.","date":"2023","source":"Cell reports","url":"https://pubmed.ncbi.nlm.nih.gov/36933219","citation_count":19,"is_preprint":false},{"pmid":"36847071","id":"PMC_36847071","title":"LncRNA LINC01871 sponging miR-142-3p to modulate ZYG11B promotes the chemoresistance of colorectal cancer cells by inducing autophagy.","date":"2023","source":"Anti-cancer drugs","url":"https://pubmed.ncbi.nlm.nih.gov/36847071","citation_count":14,"is_preprint":false},{"pmid":"40082426","id":"PMC_40082426","title":"Identification of a non-inhibitory aptameric ligand to CRL2ZYG11B E3 ligase for targeted protein degradation.","date":"2025","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/40082426","citation_count":11,"is_preprint":false},{"pmid":"35730627","id":"PMC_35730627","title":"Silencing of circular RNA‑ZYG11B exerts a neuroprotective effect against retinal neurodegeneration.","date":"2022","source":"International journal of molecular medicine","url":"https://pubmed.ncbi.nlm.nih.gov/35730627","citation_count":7,"is_preprint":false},{"pmid":"35636250","id":"PMC_35636250","title":"Structural insights into ORF10 recognition by ZYG11B.","date":"2022","source":"Biochemical and biophysical research communications","url":"https://pubmed.ncbi.nlm.nih.gov/35636250","citation_count":5,"is_preprint":false},{"pmid":"40135890","id":"PMC_40135890","title":"ZYG11B suppresses multiple enteroviruses by triggering viral VP1 degradation.","date":"2025","source":"Journal of virology","url":"https://pubmed.ncbi.nlm.nih.gov/40135890","citation_count":3,"is_preprint":false},{"pmid":"41917018","id":"PMC_41917018","title":"Structures of ZYG11B-EloB-EloC-substrate complex reveal mechanisms of CRL2ZYG11B assembly and function.","date":"2026","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/41917018","citation_count":0,"is_preprint":false},{"pmid":null,"id":"bio_10.1101_2024.06.24.600508","title":"Structure of the E3 ligase CRL2-ZYG11B with substrates reveals the molecular basis for N-degron recognition and ubiquitination","date":"2024-06-24","source":"bioRxiv","url":"https://doi.org/10.1101/2024.06.24.600508","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":7840,"output_tokens":3004,"usd":0.03429,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":10357,"output_tokens":3451,"usd":0.06903,"stage2_stop_reason":"end_turn"},"total_usd":0.10332,"stage1_batch_id":"msgbatch_01UT1AahFPCAyn2uVweS7A9z","stage2_batch_id":"msgbatch_01HuKWtKNT4Pqj1bc8eEP5RV","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2021,\n      \"finding\": \"Crystal structures of ZYG11B bound to various Gly/N-degrons reveal that ZYG11B uses its armadillo (ARM) repeats to form a deep and narrow cavity that engages mainly the first four residues of Gly/N-degrons, with the α-amino group of the degron accommodated in an acidic pocket via five conserved hydrogen bonds, establishing the structural basis for specific Gly/N-degron recognition by the CRL2ZYG11B E3 ubiquitin ligase complex.\",\n      \"method\": \"X-ray crystallography of ZYG11B bound to Gly/N-degron peptides, combined with biochemical binding assays\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structures with multiple ligands plus biochemical validation; single lab but multiple orthogonal methods\",\n      \"pmids\": [\"34214466\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"SARS-CoV-2 ORF10 physically interacts with ZYG11B (the substrate receptor of CRL2ZYG11B), and the N-terminus of ORF10 is critical for this interaction; however, ORF10 does not function to inhibit or hijack CRL2ZYG11B, and ZYG11B (and its paralog ZER1) are dispensable for SARS-CoV-2 infection in cultured cells.\",\n      \"method\": \"Co-immunoprecipitation, N-terminal deletion analysis, ZYG11B/ZER1 knockout cell infection assays\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP plus KO functional assay in a single lab, two orthogonal methods\",\n      \"pmids\": [\"33827988\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"A truncating mutation in ZYG11B (p.Glu537*) produces a protein with altered subcellular localization in HeLa cells compared to wild-type ZYG11B; knockdown of the zebrafish ZYG11B homologue disrupts craniofacial cartilage architecture and notochord development, and ZYG11B expression regulates the cartilage master regulator SOX6 and is regulated by retinoic acid.\",\n      \"method\": \"Overexpression and immunofluorescence of wild-type vs. mutant ZYG11B in HeLa cells; morpholino-based knockdown in zebrafish embryos with phenotypic readout; RT-qPCR for SOX6\",\n      \"journal\": \"Molecular genetics & genomic medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — direct localization experiment with functional consequence plus zebrafish KD phenotype, single lab, two orthogonal model systems\",\n      \"pmids\": [\"32738032\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"ZYG11B (as part of CRL2ZYG11B) recognizes not only N-terminal glycine but also small Nt-residues (Ser, Ala, Cys) in vitro; however, Nt-acetylation of Ser, Ala, and Cys by N-terminal acetyltransferases (NATs) shields these residues from ZYG11B recognition in cells, while in NAT-deficient cells ZYG11B/ZER1 targets these unacetylated small Nt-residues for proteasomal degradation. Crystal structures of ZYG11B bound to these small Nt-residues reveal the molecular mechanism of non-acetylated substrate recognition.\",\n      \"method\": \"In vitro binding assays, cellular degradation assays in NAT-deficient cells, X-ray crystallography\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structures plus in vitro binding plus cellular functional assays, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"36496439\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"ZYG11B acts as an amplifier of cGAS-mediated innate immune signaling by enhancing cGAS-DNA binding affinity, potentiating cGAS-DNA condensation, and stabilizing the cGAS-DNA condensed complex, thereby promoting cGAMP production and downstream interferon/cytokine transcription; knockdown of ZYG11B impairs these responses. Additionally, HSV-1 infection induces ZYG11B degradation in a cGAS-independent manner.\",\n      \"method\": \"ZYG11B knockdown with cGAMP measurement, co-immunoprecipitation/pulldown for cGAS-DNA-ZYG11B interaction, condensation assays, HSV-1 infection assays\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — KD with defined biochemical readout plus binding/condensation assays, single lab, multiple orthogonal methods\",\n      \"pmids\": [\"36933219\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Crystal structure of ZYG11B bound to the N-terminal peptide of SARS-CoV-2 ORF10 reveals the structural basis for recognition of the ORF10 N-terminus by ZYG11B's substrate-binding cavity.\",\n      \"method\": \"X-ray crystallography of ZYG11B–ORF10 N-terminal peptide complex\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — crystal structure from single lab, single method, no accompanying mutagenesis or functional validation reported in abstract\",\n      \"pmids\": [\"35636250\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"ZYG11B, as part of the CRL2ZYG11B complex, targets Enterovirus 71 (EV71) structural protein VP1 for proteasomal degradation via K33-linked ubiquitination; mass spectrometry and immunoprecipitation confirmed the ZYG11B–VP1 interaction and identified key domains on ZYG11B required for VP1 binding and CUL2 recruitment. ZYG11B also restricts related enteroviruses (CA6, CA16, EVD68) whose VP1 ubiquitination sites are highly conserved.\",\n      \"method\": \"Mass spectrometry, co-immunoprecipitation, domain mapping, proteasome inhibitor assays, K33 ubiquitination linkage analysis, viral replication functional assays\",\n      \"journal\": \"Journal of virology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — MS plus Co-IP plus functional viral assays in single lab, multiple orthogonal methods\",\n      \"pmids\": [\"40135890\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"Cryo-EM structures of full-length human ZYG11B in complex with EloB-EloC adaptor and a Gly/N-degron peptide reveal a seahorse-like architecture with distinct interfaces for adaptor binding and substrate engagement. ZYG11B adopts both monomeric and dimeric assemblies, with the dimer stabilizing two substrate-binding sites in opposite orientations. Functional assays demonstrate that interfaces mediating adaptor recruitment, substrate binding, and dimerization are all essential for substrate degradation.\",\n      \"method\": \"Cryo-EM structure determination of full-length ZYG11B–EloB–EloC–substrate complex; mutagenesis of assembly interfaces with substrate degradation functional assays\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — cryo-EM structures plus interface mutagenesis plus functional degradation assays, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"41917018\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Cryo-EM structures of the full CRL2-ZYG11B holoenzyme alone and in complex with a Gly/N-degron peptide from NLRP1 reveal that ZYG11B folds into a Leucine-Rich Repeat domain followed by two armadillo repeat domains that mediate CRL2 assembly and NLRP1 Gly/N-degron recognition. ZYG11B promotes NLRP1 inflammasome activation by recognizing and ubiquitinating the Gly/N-degron exposed after viral protease cleavage of NLRP1; blocking ZYG11B recognition of this degron inhibits viral protease-mediated NLRP1 inflammasome activation.\",\n      \"method\": \"Cryo-EM structure of CRL2-ZYG11B holoenzyme ± Gly/N-degron peptide; in vitro ubiquitination assays; functional inflammasome activation assays with blocking experiments\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — cryo-EM plus functional assays, but preprint with no peer review; multiple orthogonal methods in single lab\",\n      \"pmids\": [\"bio_10.1101_2024.06.24.600508\"],\n      \"is_preprint\": true\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"A non-inhibitory DNA aptamer targeting ZYG11B was identified and validated as an E3 ligase warhead for PROTAC-based targeted protein degradation (ZATAC platform), demonstrating that ZYG11B can be recruited to degrade diverse neo-substrates (nucleolin, SOX2, mutant p53-R175H) when bridged via an aptamer, and that cancer-cell-targeted 3WJ-ZATACs achieve dual-target degradation and suppress tumor growth in vivo without noticeable toxicity.\",\n      \"method\": \"Aptamer selection and binding validation; PROTAC/ZATAC functional degradation assays for multiple target proteins; in vivo tumor xenograft experiments\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — functional degradation assays with multiple substrates plus in vivo data, single lab, multiple orthogonal methods\",\n      \"pmids\": [\"40082426\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"ZYG11B is the substrate receptor of the CRL2ZYG11B E3 ubiquitin ligase complex, which uses its armadillo (and leucine-rich) repeat domains to recognize small N-terminal residues—primarily N-terminal glycine (Gly/N-degrons) but also unacetylated Ala, Ser, and Cys—via a conserved acidic pocket, targeting substrates (including NLRP1, viral VP1 proteins, and myristoylation-pathway substrates) for K33/K48-linked ubiquitination and proteasomal degradation; additionally, ZYG11B acts outside its E3 role as a positive regulator of cGAS innate immune signaling by enhancing cGAS-DNA binding and condensation, while the full CRL2ZYG11B holoenzyme assembly involves EloB/EloC adaptors and ZYG11B can form functionally relevant dimers that present two substrate-binding sites.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"ZYG11B is the substrate-recognition subunit of the CRL2ZYG11B E3 ubiquitin ligase, which targets proteins bearing small N-terminal residues for proteasomal degradation through the N-degron pathway [#0, #8]. Its armadillo repeats form a deep, narrow cavity whose acidic pocket coordinates the α-amino group of an N-terminal glycine through five conserved hydrogen bonds, accounting for selective Gly/N-degron recognition; the same site also engages unacetylated Ser, Ala, and Cys, residues that N-terminal acetyltransferases normally shield from ZYG11B in cells [#0, #3]. Full-length ZYG11B folds into a leucine-rich repeat domain followed by two armadillo domains and assembles into the holoenzyme via EloB/EloC adaptors and CUL2, with adaptor-binding, substrate-binding, and dimerization interfaces all required for substrate degradation; ZYG11B can dimerize to present two substrate sites [#7, #8]. Through this activity it ubiquitinates physiological and pathogen-derived substrates: it promotes NLRP1 inflammasome activation by recognizing the Gly/N-degron exposed after viral protease cleavage of NLRP1 [#8], and it restricts enteroviruses by K33-linked ubiquitination of the structural protein VP1 [#6]. Independently of its ligase role, ZYG11B amplifies cGAS-mediated innate immune signaling by enhancing cGAS–DNA binding and condensation to potentiate cGAMP production [#4]. ZYG11B has also been repurposed as an E3 warhead for aptamer-bridged targeted protein degradation of neo-substrates [#9].\",\n  \"teleology\": [\n    {\n      \"year\": 2020,\n      \"claim\": \"Before its biochemical role was defined, ZYG11B was linked to vertebrate development, establishing a phenotypic consequence of disrupting the gene.\",\n      \"evidence\": \"Mutant-vs-wild-type localization in HeLa cells plus morpholino knockdown in zebrafish with SOX6 expression readout\",\n      \"pmids\": [\"32738032\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Does not connect the developmental phenotype to E3 ligase activity or specific substrates\", \"Morpholino knockdown lacks genetic rescue\", \"Truncating-mutation localization effect not mechanistically explained\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Crystal structures defined how ZYG11B reads an N-terminal glycine, establishing the structural basis of Gly/N-degron recognition by CRL2ZYG11B.\",\n      \"evidence\": \"X-ray crystallography of ZYG11B–Gly/N-degron peptide complexes with biochemical binding assays\",\n      \"pmids\": [\"34214466\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Used isolated armadillo-domain structures rather than the full holoenzyme\", \"Did not address physiological substrate identity\", \"Catalytic ubiquitin transfer not visualized\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"A test of whether SARS-CoV-2 ORF10 hijacks CRL2ZYG11B showed physical interaction via the ORF10 N-terminus but no functional requirement, ruling out a ubiquitin-ligase-hijacking model.\",\n      \"evidence\": \"Reciprocal Co-IP, N-terminal deletion analysis, and ZYG11B/ZER1 knockout infection assays\",\n      \"pmids\": [\"33827988\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Does not explain why ORF10 binds ZYG11B if not to modulate it\", \"Negative infection result confined to cultured cells\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Extending the degron repertoire, ZYG11B was shown to recognize unacetylated Ser/Ala/Cys N-termini, integrating N-terminal acetylation status into substrate selection.\",\n      \"evidence\": \"In vitro binding, degradation assays in NAT-deficient cells, and X-ray crystallography of small-Nt-residue complexes\",\n      \"pmids\": [\"36496439\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Endogenous substrates exposed in NAT-deficient states not enumerated\", \"Relative in vivo contribution versus paralog ZER1 not resolved\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"A co-crystal structure detailed how ZYG11B's substrate cavity engages the ORF10 N-terminal peptide, structurally rationalizing the earlier interaction.\",\n      \"evidence\": \"X-ray crystallography of the ZYG11B–ORF10 N-terminal peptide complex\",\n      \"pmids\": [\"35636250\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No accompanying mutagenesis or functional validation\", \"Does not reconcile binding with the lack of functional consequence for infection\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"ZYG11B was found to act outside its E3 role as a positive regulator of cGAS, revealing a moonlighting function in innate immune signaling.\",\n      \"evidence\": \"ZYG11B knockdown with cGAMP measurement, cGAS-DNA-ZYG11B pulldown, condensation assays, and HSV-1 infection assays\",\n      \"pmids\": [\"36933219\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism linking ZYG11B binding to condensate stabilization not structurally defined\", \"Whether ligase activity contributes to cGAS regulation unresolved\", \"HSV-1-induced ZYG11B degradation pathway unidentified\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Holoenzyme cryo-EM with an NLRP1 degron tied ZYG11B's degron recognition to inflammasome biology, showing it ubiquitinates the Gly/N-degron exposed by viral protease cleavage of NLRP1.\",\n      \"evidence\": \"Cryo-EM of CRL2-ZYG11B ± NLRP1 Gly/N-degron peptide, in vitro ubiquitination, and inflammasome activation/blocking assays (preprint)\",\n      \"pmids\": [\"bio_10.1101_2024.06.24.600508\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Preprint without peer review\", \"Physiological breadth of NLRP1 regulation in primary immune cells not established\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"ZYG11B was shown to restrict enteroviruses by K33-linked ubiquitination of VP1, demonstrating antiviral substrate targeting and mapping the required ZYG11B domains.\",\n      \"evidence\": \"Mass spectrometry, Co-IP, domain mapping, proteasome inhibitor and K33-linkage analysis, and viral replication assays\",\n      \"pmids\": [\"40135890\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"In vivo antiviral relevance not tested\", \"Why K33 rather than K48 linkage is used mechanistically unclear\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"A non-inhibitory aptamer warhead established ZYG11B as a recruitable E3 for targeted protein degradation, enabling neo-substrate destruction in vivo.\",\n      \"evidence\": \"Aptamer selection, ZATAC degradation assays for nucleolin/SOX2/p53-R175H, and tumor xenograft experiments\",\n      \"pmids\": [\"40082426\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Endogenous-substrate physiology not addressed by this engineering study\", \"Long-term specificity and toxicity beyond xenografts unknown\"]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"Full-length cryo-EM resolved how ZYG11B assembles with EloB/EloC and dimerizes, establishing the complete architecture required for substrate degradation.\",\n      \"evidence\": \"Cryo-EM of full-length ZYG11B–EloB–EloC–substrate complex with interface mutagenesis and degradation assays\",\n      \"pmids\": [\"41917018\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional advantage of dimer over monomer in cells not quantified\", \"Dynamics of substrate hand-off to the catalytic module not captured\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How ZYG11B's ligase-dependent N-degron functions and its ligase-independent cGAS regulation are coordinated, and the full endogenous substrate set, remain open.\",\n      \"evidence\": null,\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No comprehensive endogenous substrate catalog\", \"Relationship between developmental phenotypes and defined substrates unestablished\", \"Division of labor with paralog ZER1 unresolved\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [6, 8]},\n      {\"term_id\": \"GO:0016874\", \"supporting_discovery_ids\": [6, 8]},\n      {\"term_id\": \"GO:0060089\", \"supporting_discovery_ids\": [0, 3]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [4]}\n    ],\n    \"localization\": [],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [0, 3, 7]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [4, 6, 8]}\n    ],\n    \"complexes\": [\"CRL2ZYG11B\"],\n    \"partners\": [\"CUL2\", \"EloB\", \"EloC\", \"cGAS\", \"NLRP1\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":6,"faith_pct":100.0}}