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- PDB-7jwb: SARS CoV2 Spike ectodomain with engineered trimerized VH binder -

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Basic information

Entry
Database: PDB / ID: 7jwb
TitleSARS CoV2 Spike ectodomain with engineered trimerized VH binder
Components
  • Spike glycoprotein
  • autonomous human heavy chain variable domain
KeywordsVIRAL PROTEIN/IMMUNE SYSTEM / Spike / VH / SARS / CoV2 / VIRAL PROTEIN-IMMUNE SYSTEM complex
Function / homology
Function and homology information


Maturation of spike protein / viral translation / Translation of Structural Proteins / Virion Assembly and Release / host cell surface / host extracellular space / symbiont-mediated-mediated suppression of host tetherin activity / Induction of Cell-Cell Fusion / structural constituent of virion / entry receptor-mediated virion attachment to host cell ...Maturation of spike protein / viral translation / Translation of Structural Proteins / Virion Assembly and Release / host cell surface / host extracellular space / symbiont-mediated-mediated suppression of host tetherin activity / Induction of Cell-Cell Fusion / structural constituent of virion / entry receptor-mediated virion attachment to host cell / membrane fusion / host cell endoplasmic reticulum-Golgi intermediate compartment membrane / Attachment and Entry / positive regulation of viral entry into host cell / receptor-mediated virion attachment to host cell / host cell surface receptor binding / symbiont-mediated suppression of host innate immune response / receptor ligand activity / endocytosis involved in viral entry into host cell / fusion of virus membrane with host plasma membrane / fusion of virus membrane with host endosome membrane / viral envelope / virion attachment to host cell / SARS-CoV-2 activates/modulates innate and adaptive immune responses / host cell plasma membrane / virion membrane / identical protein binding / membrane / plasma membrane
Similarity search - Function
Spike (S) protein S1 subunit, receptor-binding domain, SARS-CoV-2 / Spike (S) protein S1 subunit, N-terminal domain, SARS-CoV-like / Coronavirus spike glycoprotein S1, C-terminal / Coronavirus spike glycoprotein S1, C-terminal / Spike glycoprotein, betacoronavirus / Spike glycoprotein, N-terminal domain superfamily / Betacoronavirus spike (S) glycoprotein S1 subunit N-terminal (NTD) domain profile. / Betacoronavirus spike (S) glycoprotein S1 subunit C-terminal (CTD) domain profile. / Spike (S) protein S1 subunit, receptor-binding domain, betacoronavirus / Spike S1 subunit, receptor binding domain superfamily, betacoronavirus ...Spike (S) protein S1 subunit, receptor-binding domain, SARS-CoV-2 / Spike (S) protein S1 subunit, N-terminal domain, SARS-CoV-like / Coronavirus spike glycoprotein S1, C-terminal / Coronavirus spike glycoprotein S1, C-terminal / Spike glycoprotein, betacoronavirus / Spike glycoprotein, N-terminal domain superfamily / Betacoronavirus spike (S) glycoprotein S1 subunit N-terminal (NTD) domain profile. / Betacoronavirus spike (S) glycoprotein S1 subunit C-terminal (CTD) domain profile. / Spike (S) protein S1 subunit, receptor-binding domain, betacoronavirus / Spike S1 subunit, receptor binding domain superfamily, betacoronavirus / Betacoronavirus spike glycoprotein S1, receptor binding / Spike glycoprotein S1, N-terminal domain, betacoronavirus-like / Betacoronavirus-like spike glycoprotein S1, N-terminal / Spike glycoprotein S2, coronavirus, heptad repeat 1 / Spike glycoprotein S2, coronavirus, heptad repeat 2 / Coronavirus spike (S) glycoprotein S2 subunit heptad repeat 1 (HR1) region profile. / Coronavirus spike (S) glycoprotein S2 subunit heptad repeat 2 (HR2) region profile. / Spike glycoprotein S2 superfamily, coronavirus / Spike glycoprotein S2, coronavirus / Coronavirus spike glycoprotein S2
Similarity search - Domain/homology
Biological speciesHomo sapiens (human)
Severe acute respiratory syndrome coronavirus 2
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 6 Å
AuthorsQCRG Structural Biology Consortium
Funding support United States, 4items
OrganizationGrant numberCountry
Defense Advanced Research Projects Agency (DARPA)HR0011-19-2-0020 United States
Quantitative Biosciences Institute United States
FastGrants United States
Laboratory for Genomics Research (LGR)Excellence in Research Award (ERA) United States
Citation
Journal: Nat Chem Biol / Year: 2021
Title: Bi-paratopic and multivalent VH domains block ACE2 binding and neutralize SARS-CoV-2.
Authors: Colton J Bracken / Shion A Lim / Paige Solomon / Nicholas J Rettko / Duy P Nguyen / Beth Shoshana Zha / Kaitlin Schaefer / James R Byrnes / Jie Zhou / Irene Lui / Jia Liu / Katarina Pance / ...Authors: Colton J Bracken / Shion A Lim / Paige Solomon / Nicholas J Rettko / Duy P Nguyen / Beth Shoshana Zha / Kaitlin Schaefer / James R Byrnes / Jie Zhou / Irene Lui / Jia Liu / Katarina Pance / / Xin X Zhou / Kevin K Leung / James A Wells /
Abstract: Neutralizing agents against SARS-CoV-2 are urgently needed for the treatment and prophylaxis of COVID-19. Here, we present a strategy to rapidly identify and assemble synthetic human variable heavy ...Neutralizing agents against SARS-CoV-2 are urgently needed for the treatment and prophylaxis of COVID-19. Here, we present a strategy to rapidly identify and assemble synthetic human variable heavy (VH) domains toward neutralizing epitopes. We constructed a VH-phage library and targeted the angiotensin-converting enzyme 2 (ACE2) binding interface of the SARS-CoV-2 Spike receptor-binding domain (Spike-RBD). Using a masked selection approach, we identified VH binders to two non-overlapping epitopes and further assembled these into multivalent and bi-paratopic formats. These VH constructs showed increased affinity to Spike (up to 600-fold) and neutralization potency (up to 1,400-fold) on pseudotyped SARS-CoV-2 virus when compared to standalone VH domains. The most potent binder, a trivalent VH, neutralized authentic SARS-CoV-2 with a half-maximal inhibitory concentration (IC) of 4.0 nM (180 ng ml). A cryo-EM structure of the trivalent VH bound to Spike shows each VH domain engaging an RBD at the ACE2 binding site, confirming our original design strategy.
#1: Journal: bioRxiv / Year: 2020
Title: Bi-paratopic and multivalent human VH domains neutralize SARS-CoV-2 by targeting distinct epitopes within the ACE2 binding interface of Spike.
Authors: Colton J Bracken / Shion A Lim / Paige Solomon / Nicholas J Rettko / Duy P Nguyen / Beth Shoshana Zha / Kaitlin Schaefer / James R Byrnes / Jie Zhou / Irene Lui / Jia Liu / Katarina Pance / ...Authors: Colton J Bracken / Shion A Lim / Paige Solomon / Nicholas J Rettko / Duy P Nguyen / Beth Shoshana Zha / Kaitlin Schaefer / James R Byrnes / Jie Zhou / Irene Lui / Jia Liu / Katarina Pance / / Xin X Zhou / Kevin K Leung / James A Wells /
Abstract: Neutralizing agents against SARS-CoV-2 are urgently needed for treatment and prophylaxis of COVID-19. Here, we present a strategy to rapidly identify and assemble synthetic human variable heavy (VH) ...Neutralizing agents against SARS-CoV-2 are urgently needed for treatment and prophylaxis of COVID-19. Here, we present a strategy to rapidly identify and assemble synthetic human variable heavy (VH) domain binders with high affinity toward neutralizing epitopes without the need for high-resolution structural information. We constructed a VH-phage library and targeted a known neutralizing site, the angiotensin-converting enzyme 2 (ACE2) binding interface of the trimeric SARS-CoV-2 Spike receptor-binding domain (Spike-RBD). Using a masked selection approach, we identified 85 unique VH binders to two non-overlapping epitopes within the ACE2 binding site on Spike-RBD. This enabled us to systematically link these VH domains into multivalent and bi-paratopic formats. These multivalent and bi-paratopic VH constructs showed a marked increase in affinity to Spike (up to 600-fold) and neutralization potency (up to 1400-fold) on pseudotyped SARS-CoV-2 virus when compared to the standalone VH domains. The most potent binder, a trivalent VH, neutralized authentic SARS-CoV-2 with half-minimal inhibitory concentration (IC ) of 4.0 nM (180 ng/mL). A cryo-EM structure of the trivalent VH bound to Spike shows each VH domain bound an RBD at the ACE2 binding site, explaining its increased neutralization potency and confirming our original design strategy. Our results demonstrate that targeted selection and engineering campaigns using a VH-phage library can enable rapid assembly of highly avid and potent molecules towards therapeutically important protein interfaces.
History
DepositionAug 25, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Oct 7, 2020Provider: repository / Type: Initial release
Revision 1.1Oct 14, 2020Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year
Revision 1.2Dec 16, 2020Group: Database references / Category: citation / citation_author
Revision 1.3Dec 30, 2020Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.year / _citation_author.identifier_ORCID
Revision 1.4Nov 13, 2024Group: Data collection / Database references ...Data collection / Database references / Refinement description / Structure summary
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / citation / database_2 / em_3d_fitting_list / em_admin / pdbx_entry_details / pdbx_initial_refinement_model / pdbx_modification_feature
Item: _citation.journal_id_ISSN / _database_2.pdbx_DOI ..._citation.journal_id_ISSN / _database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type / _em_admin.last_update

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Structure visualization

Movie
  • Deposited structure unit
  • Imaged by Jmol
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  • Superimposition on EM map
  • EMDB-22514
  • Imaged by UCSF Chimera
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Structure viewerMolecule:
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Assembly

Deposited unit
D: autonomous human heavy chain variable domain
A: Spike glycoprotein
B: Spike glycoprotein
C: Spike glycoprotein


Theoretical massNumber of molelcules
Total (without water)445,5244
Polymers445,5244
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area28730 Å2
ΔGint-126 kcal/mol
Surface area139090 Å2

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Components

#1: Antibody autonomous human heavy chain variable domain


Mass: 44264.402 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Escherichia coli K-12 (bacteria)
#2: Protein Spike glycoprotein / S glycoprotein / E2 / Peplomer protein


Mass: 133753.250 Da / Num. of mol.: 3 / Mutation: R682G,R683S,R685S,R986P,V987P
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Severe acute respiratory syndrome coronavirus 2
Gene: S, 2 / Production host: Cricetulus griseus (Chinese hamster) / References: UniProt: P0DTC2
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

Component
IDNameTypeEntity IDParent-IDSource
1Complex of SARS CoV2 Spike ectodomain with engineered trimerized VH domainCOMPLEXall0MULTIPLE SOURCES
2engineered trimerized VH domainCOMPLEX#11RECOMBINANT
3SARS CoV2 Spike ectodomainCOMPLEX#21RECOMBINANT
Molecular weightValue: 0.445 MDa / Experimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Homo sapiens (human)9606
23Severe acute respiratory syndrome coronavirus 22697049
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
12Escherichia coli K-12 (bacteria)83333
23Cricetulus griseus (Chinese hamster)10029
Buffer solutionpH: 8 / Details: 20 mM HEPES, pH 8, 200 mM NaCl
SpecimenConc.: 0.9 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 200 divisions/in. / Grid type: UltrAuFoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD
Image recordingElectron dose: 78 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

EM softwareName: UCSF Chimera / Category: model fitting
Image processing
IDImage recording-ID
11
21
CTF correction
IDEM image processing-IDType
11PHASE FLIPPING AND AMPLITUDE CORRECTION
22PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstruction

Entry-ID: 7JWB / Num. of particles: 21000 / Symmetry type: POINT

IDResolution (Å)Resolution methodImage processing-IDDetails
16OTHER1This reconstruction is a Gaussian low pass filtered map of a 3.2A gold standard FSC map, to better account for the VH density. The model was fit and relaxed into this map.
23.2FSC 0.143 CUT-OFF2Same reconstruction as above but filtered based on the gold standard FSC to 3.2A. Shows high resolution features but density for the low occupancy VH is poorly resolved.
Atomic model buildingProtocol: RIGID BODY FIT / Target criteria: cross correlation
Atomic model building

3D fitting-ID: 1 / Source name: PDB / Type: experimental model

IDPDB-IDPdb chain-IDAccession codeInitial refinement model-IDPdb chain residue range
16X2B

6x2b

6X2B1
24G80

4g80

A4G8021-124

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