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- PDB-9cgv: SARS-CoV-2 nsp12 NiRAN domain bound to a covalent inhibitor SW090466-1 -

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Basic information

Entry
Database: PDB / ID: 9cgv
TitleSARS-CoV-2 nsp12 NiRAN domain bound to a covalent inhibitor SW090466-1
Components
  • (Non-structural protein ...) x 2
  • RNA primer
  • RNA template
  • RNA-directed RNA polymerase nsp12
KeywordsVIRAL PROTEIN/RNA/INHIBITOR / Complex / Covalent / Inhibitor / SARS-CoV-2 / Polymerase / NiRAN / VIRAL PROTEIN-RNA-INHIBITOR complex
Function / homology
Function and homology information


protein guanylyltransferase activity / RNA endonuclease activity producing 3'-phosphomonoesters, hydrolytic mechanism / mRNA guanylyltransferase activity / 5'-3' RNA helicase activity / Lyases; Phosphorus-oxygen lyases / Assembly of the SARS-CoV-2 Replication-Transcription Complex (RTC) / symbiont-mediated suppression of host cytoplasmic pattern recognition receptor signaling pathway via inhibition of TBK1 activity / Maturation of replicase proteins / TRAF3-dependent IRF activation pathway / ISG15-specific peptidase activity ...protein guanylyltransferase activity / RNA endonuclease activity producing 3'-phosphomonoesters, hydrolytic mechanism / mRNA guanylyltransferase activity / 5'-3' RNA helicase activity / Lyases; Phosphorus-oxygen lyases / Assembly of the SARS-CoV-2 Replication-Transcription Complex (RTC) / symbiont-mediated suppression of host cytoplasmic pattern recognition receptor signaling pathway via inhibition of TBK1 activity / Maturation of replicase proteins / TRAF3-dependent IRF activation pathway / ISG15-specific peptidase activity / Transcription of SARS-CoV-2 sgRNAs / Translation of Replicase and Assembly of the Replication Transcription Complex / snRNP Assembly / Replication of the SARS-CoV-2 genome / Hydrolases; Acting on ester bonds; Exoribonucleases producing 5'-phosphomonoesters / host cell endoplasmic reticulum-Golgi intermediate compartment / double membrane vesicle viral factory outer membrane / SARS coronavirus main proteinase / 5'-3' DNA helicase activity / 3'-5'-RNA exonuclease activity / host cell endosome / symbiont-mediated degradation of host mRNA / mRNA guanylyltransferase / symbiont-mediated suppression of host ISG15-protein conjugation / G-quadruplex RNA binding / symbiont-mediated suppression of host toll-like receptor signaling pathway / symbiont-mediated suppression of host cytoplasmic pattern recognition receptor signaling pathway via inhibition of IRF3 activity / omega peptidase activity / mRNA (guanine-N7)-methyltransferase / SARS-CoV-2 modulates host translation machinery / methyltransferase cap1 / host cell Golgi apparatus / symbiont-mediated suppression of host NF-kappaB cascade / symbiont-mediated perturbation of host ubiquitin-like protein modification / DNA helicase / methyltransferase cap1 activity / ubiquitinyl hydrolase 1 / cysteine-type deubiquitinase activity / mRNA 5'-cap (guanine-N7-)-methyltransferase activity / Hydrolases; Acting on peptide bonds (peptidases); Cysteine endopeptidases / single-stranded RNA binding / regulation of autophagy / host cell perinuclear region of cytoplasm / viral protein processing / lyase activity / host cell endoplasmic reticulum membrane / RNA helicase / symbiont-mediated suppression of host type I interferon-mediated signaling pathway / symbiont-mediated suppression of host gene expression / copper ion binding / viral translational frameshifting / symbiont-mediated activation of host autophagy / RNA-directed RNA polymerase / cysteine-type endopeptidase activity / viral RNA genome replication / RNA-directed RNA polymerase activity / DNA-templated transcription / lipid binding / host cell nucleus / SARS-CoV-2 activates/modulates innate and adaptive immune responses / ATP hydrolysis activity / proteolysis / RNA binding / zinc ion binding / ATP binding / membrane
Similarity search - Function
RNA-dependent RNA polymerase, SARS-CoV-like / Nonstructural protein 14, betacoronavirus / NSP15, NendoU domain, coronavirus / Nonstructural protein 15, middle domain, alpha/betacoronavirus / Nonstructural protein 15, N-terminal domain, alpha/beta-coronavirus / NSP14, guanine-N7-methyltransferase domain, coronavirus / Coronavirus (CoV) guanine-N7-methyltransferase (N7-MTase) domain profile. / Coronavirus (CoV) Nsp15 N-terminal oligomerization domain profile. / NSP12 RNA-dependent RNA polymerase, coronavirus / : ...RNA-dependent RNA polymerase, SARS-CoV-like / Nonstructural protein 14, betacoronavirus / NSP15, NendoU domain, coronavirus / Nonstructural protein 15, middle domain, alpha/betacoronavirus / Nonstructural protein 15, N-terminal domain, alpha/beta-coronavirus / NSP14, guanine-N7-methyltransferase domain, coronavirus / Coronavirus (CoV) guanine-N7-methyltransferase (N7-MTase) domain profile. / Coronavirus (CoV) Nsp15 N-terminal oligomerization domain profile. / NSP12 RNA-dependent RNA polymerase, coronavirus / : / Coronavirus Nsp12 RNA-dependent RNA polymerase (RdRp) domain profile. / Coronavirus Nsp12 Interface domain profile. / : / : / Coronavirus Nonstructural protein 13, 1B domain / Coronavirus Non-structural protein 13, zinc-binding domain / Coronavirus Nonstructural protein 13, stalk domain / Nidovirus 2-O-methyltransferase / Nidovirus 2'-O-methyltransferase (2'-O-MTase) domain profile. / Non-structural protein NSP15, N-terminal domain superfamily, coronavirus / Non-structural protein NSP15, middle domain superfamily / Coronavirus replicase NSP15, N-terminal oligomerization / Nonstructural protein 15, middle domain, coronavirus / Nonstructural protein 13, 1B domain, coronavirus / Coronavirus replicase NSP15, middle domain / Coronavirus replicase NSP15, N-terminal oligomerisation / Nidovirus 3'-5' exoribonuclease domain / Nidovirus 3'-5' exoribonuclease (ExoN) domain profile. / Non-structural protein NSP16, coronavirus-like / Non-structural protein 14, coronavirus / RNA polymerase, N-terminal, coronavirus / Coronavirus 2'-O-methyltransferase / Coronavirus proofreading exoribonuclease / Coronavirus RNA-dependent RNA polymerase, N-terminal / Nonstructural protein 13, zinc-binding domain, coronavirus-like / Coronaviridae zinc-binding (CV ZBD) domain profile. / Arterivirus Nsp11 N-terminal/Coronavirus NSP15 middle domain / Arterivirus Nsp11 N-terminal/coronavirus NSP15 middle (AV-Nsp11N/CoV-Nsp15M) domain profile. / Nidoviral uridylate-specific endoribonuclease (NendoU) domain profile. / Nidovirus RdRp-associated nucleotidyl transferase (NiRAN) domain / Nidovirus RdRp-associated nucleotidyl transferase (NiRAN) domain profile. / Endoribonuclease EndoU-like / NendoU domain, nidovirus / Coronavirus replicase NSP15, uridylate-specific endoribonuclease / : / Lipocalin signature. / DNA2/NAM7 helicase-like, C-terminal / AAA domain / (+) RNA virus helicase core domain / (+)RNA virus helicase core domain profile. / Non-structural protein NSP3, SUD-N (Mac2) domain, betacoronavirus / Sarbecovirus Nsp3c-N domain profile. / Non-structural protein NSP3, N-terminal, betacoronavirus / Polyprotein cleavage domain PL2pro superfamily, betacoronavirus / Non-structural protein NSP3, SUD-N (Mac2) domain superfamily, betacoronavirus / Betacoronavirus SUD-C domain / Betacoronavirus replicase NSP3, N-terminal / NSP1 globular domain superfamily, betacoronavirus / Non-structural protein 2, SARS-CoV-like / Carbamoyl-phosphate synthase subdomain signature 2. / Betacoronavirus Nsp3c-M domain profile. / NSP1, globular domain, betacoronavirus / Non-structural protein NSP3, SUD-M domain, betacoronavirus / Non-structural protein NSP3, SUD-M domain superfamily, betacoronavirus / Betacoronavirus replicase NSP1 / Betacoronavirus single-stranded poly(A) binding domain / NSP1, C-terminal domain, betacoronavirus / : / Betacoronavirus (BetaCoV) Nsp1 C-terminal domain profile. / Betacoronavirus Nsp3e group 2-specific marker (G2M) domain profile. / Betacoronavirus Nsp3c-C domain profile. / Betacoronavirus Nsp3e nucleic acid-binding (NAB) domain profile. / DPUP/SUD, C-terminal, betacoronavirus / Non-structural protein NSP3, nucleic acid-binding domain, betacoronavirus / Non-structural protein NSP3A domain-like superfamily / Non-structural protein NSP3, nucleic acid-binding domain superfamily, betacoronavirus / Non-structural protein 6, betacoronavirus / Betacoronavirus nucleic acid-binding (NAB) / Papain-like viral protease, palm and finger domains, coronavirus / Papain-like protease, N-terminal domain superfamily, coronavirus / Coronavirus replicase NSP2, N-terminal / : / Coronavirus replicase NSP2, C-terminal / Coronavirus (CoV) Nsp2 middle domain profile. / NSP1, globular domain, alpha/betacoronavirus / Coronavirus (CoV) Nsp1 globular domain profile. / Coronavirus (CoV) Nsp2 N-terminal domain profile. / Coronavirus (CoV) Nsp2 C-terminal domain profile. / Nonstructural protein 2, N-terminal domain, coronavirus / Non-structural protein 2, C-terminal domain, coronavirus / NSP3, second ubiquitin-like (Ubl) domain, coronavirus / Coronavirus Nsp3a Ubl domain profile. / Coronavirus Nsp3d Ubl domain profile. / NSP3, first ubiquitin-like (Ubl) domain, coronavirus / Peptidase family C16 domain profile. / : / : / Coronavirus replicase NSP7 / Coronavirus 3Ecto domain profile. / Coronavirus RNA-dependent RNA polymerase (RdRp) Nsp7 cofactor domain profile.
Similarity search - Domain/homology
: / : / RNA / RNA (> 10) / Replicase polyprotein 1ab
Similarity search - Component
Biological speciesSevere acute respiratory syndrome coronavirus 2
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.7 Å
AuthorsOsinski, A. / Hernandez, G. / Tagliabracci, V.S.
Funding support United States, 5items
OrganizationGrant numberCountry
Howard Hughes Medical Institute (HHMI) United States
W. M. Keck Foundation United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM135189 United States
Welch FoundationI-1911 United States
Life Sciences Research Foundation United States
CitationJournal: J Biol Chem / Year: 2025
Title: Covalent inhibition of the SARS-CoV-2 NiRAN domain via an active-site cysteine.
Authors: Genaro Hernandez / Adam Osinski / Abir Majumdar / Jennifer L Eitson / Monika Antczak / Krzysztof Pawłowski / Hanspeter Niederstrasser / Kelly A Servage / Bruce Posner / John W Schoggins / ...Authors: Genaro Hernandez / Adam Osinski / Abir Majumdar / Jennifer L Eitson / Monika Antczak / Krzysztof Pawłowski / Hanspeter Niederstrasser / Kelly A Servage / Bruce Posner / John W Schoggins / Joseph M Ready / Vincent S Tagliabracci /
Abstract: The kinase-like nidovirus RdRp-associated nucleotidyl transferase (NiRAN) domain of nsp12 in SARS-CoV-2 catalyzes the formation of the 5' RNA cap structure. This activity is required for viral ...The kinase-like nidovirus RdRp-associated nucleotidyl transferase (NiRAN) domain of nsp12 in SARS-CoV-2 catalyzes the formation of the 5' RNA cap structure. This activity is required for viral replication, offering a new target for the development of antivirals. Here, we develop a high-throughput assay to screen for small molecule inhibitors targeting the SARS-CoV-2 NiRAN domain. We identified NiRAN covalent inhibitor 2 (NCI-2), a compound with a reactive chloromethyl group that covalently binds to an active site cysteine (Cys53) in the NiRAN domain, inhibiting its activity. NCI-2 can enter cells, bind to, and inactivate ectopically expressed nsp12. A cryo-EM reconstruction of the SARS-CoV-2 replication-transcription complex bound to NCI-2 offers a detailed structural blueprint for rational drug design. Although NCI-2 showed limited potency against SARS-CoV-2 replication in cells, our work lays the groundwork for developing more potent and selective inhibitors targeting the NiRAN domain. This approach presents a promising therapeutic strategy for effectively combating COVID-19 and potentially mitigating future coronavirus outbreaks.
History
DepositionJul 1, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Mar 12, 2025Provider: repository / Type: Initial release
Revision 1.1Mar 19, 2025Group: Data collection / Database references / Category: citation / em_admin
Item: _citation.page_last / _citation.pdbx_database_id_PubMed ..._citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title / _em_admin.last_update
Revision 1.2Apr 23, 2025Group: Data collection / Database references / Category: citation / em_admin / Item: _citation.journal_volume / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: RNA-directed RNA polymerase nsp12
B: Non-structural protein 8
C: Non-structural protein 7
D: Non-structural protein 8
P: RNA primer
T: RNA template
hetero molecules


Theoretical massNumber of molelcules
Total (without water)191,01410
Polymers190,6216
Non-polymers3934
Water4,648258
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Protein , 1 types, 1 molecules A

#1: Protein RNA-directed RNA polymerase nsp12


Mass: 108767.094 Da / Num. of mol.: 1 / Fragment: UNP residues 4392-5324, fused to 6xHis-TEV
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Severe acute respiratory syndrome coronavirus 2
Gene: rep, 1a-1b / Cell line (production host): Sf21 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P0DTD1

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Non-structural protein ... , 2 types, 3 molecules BDC

#2: Protein Non-structural protein 8 / nsp8


Mass: 21903.047 Da / Num. of mol.: 2 / Fragment: UNP residues 3943-4140
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Severe acute respiratory syndrome coronavirus 2
Gene: rep, 1a-1b / Production host: Escherichia coli (E. coli) / Strain (production host): ROSETTA / References: UniProt: P0DTD1
#3: Protein Non-structural protein 7 / nsp7


Mass: 9248.804 Da / Num. of mol.: 1 / Fragment: UNP residues 3860-3942
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Severe acute respiratory syndrome coronavirus 2
Gene: rep, 1a-1b / Production host: Escherichia coli (E. coli) / Strain (production host): ROSETTA / References: UniProt: P0DTD1

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RNA chain , 2 types, 2 molecules PT

#4: RNA chain RNA primer


Mass: 11157.644 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#5: RNA chain RNA template


Mass: 17641.477 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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Non-polymers , 4 types, 262 molecules

#6: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Zn
#7: Chemical ChemComp-A1AWQ / methyl (8S)-7-hydroxy-5-methylpyrazolo[1,5-a]pyrimidine-3-carboxylate


Mass: 207.186 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C9H9N3O3 / Feature type: SUBJECT OF INVESTIGATION
#8: Chemical ChemComp-MN / MANGANESE (II) ION


Mass: 54.938 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mn
#9: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 258 / Source method: isolated from a natural source / Formula: H2O

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Details

Has ligand of interestY
Has protein modificationY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: SARS-CoV-2 nsp12 NiRAN domain bound to a covalent inhibitor SW090466-1
Type: COMPLEX / Entity ID: #1-#5 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Severe acute respiratory syndrome coronavirus 2
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm) / Strain: Sf21
Buffer solutionpH: 8
SpecimenConc.: 3.6 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2400 nm / Nominal defocus min: 1100 nm
Image recordingElectron dose: 60 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

EM software
IDNameVersionCategory
2PHENIX1.21_5207model refinement
3SerialEMimage acquisition
5cryoSPARCCTF correction
10cryoSPARCinitial Euler assignment
11cryoSPARCfinal Euler assignment
12RELIONclassification
13cryoSPARC3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 77971 / Symmetry type: POINT
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 48.09 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.002710427
ELECTRON MICROSCOPYf_angle_d0.454114278
ELECTRON MICROSCOPYf_chiral_restr0.03751619
ELECTRON MICROSCOPYf_plane_restr0.00291711
ELECTRON MICROSCOPYf_dihedral_angle_d14.31533970

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