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- PDB-8elq: Crystal structure of SARS-CoV-2 spike protein receptor-binding do... -

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Basic information

Entry
Database: PDB / ID: 8elq
TitleCrystal structure of SARS-CoV-2 spike protein receptor-binding domain in complex with antibody CC12.1 Fab and nanobody Nb-C4-255
Components
  • CC12.1 Fab heavy chain
  • CC12.1 Fab light chain
  • Nanobody Nb-C4-255
  • Spike protein S1
KeywordsVIRAL PROTEIN/IMMUNE SYSTEM / synthetic / nanobody / SARS-CoV-2 / coronavirus / neutralization / IMMUNE SYSTEM / 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 / suppression by virus of host tetherin activity / Induction of Cell-Cell Fusion / structural constituent of virion / host cell endoplasmic reticulum-Golgi intermediate compartment membrane ...Maturation of spike protein / viral translation / Translation of Structural Proteins / Virion Assembly and Release / host cell surface / host extracellular space / suppression by virus of host tetherin activity / Induction of Cell-Cell Fusion / structural constituent of virion / host cell endoplasmic reticulum-Golgi intermediate compartment membrane / entry receptor-mediated virion attachment to host cell / receptor-mediated endocytosis of virus by host cell / Attachment and Entry / membrane fusion / positive regulation of viral entry into host cell / receptor-mediated virion attachment to host cell / receptor ligand activity / host cell surface receptor binding / 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 / membrane / identical protein binding / 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 / Betacoronavirus spike (S) glycoprotein S1 subunit N-terminal (NTD) domain profile. / Spike glycoprotein, N-terminal domain superfamily / Betacoronavirus spike (S) glycoprotein S1 subunit C-terminal (CTD) domain profile. / Spike glycoprotein, betacoronavirus / 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 ...Spike (S) protein S1 subunit, receptor-binding domain, SARS-CoV-2 / Spike (S) protein S1 subunit, N-terminal domain, SARS-CoV-like / Betacoronavirus spike (S) glycoprotein S1 subunit N-terminal (NTD) domain profile. / Spike glycoprotein, N-terminal domain superfamily / Betacoronavirus spike (S) glycoprotein S1 subunit C-terminal (CTD) domain profile. / Spike glycoprotein, betacoronavirus / 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 2 (HR2) region profile. / Coronavirus spike (S) glycoprotein S2 subunit heptad repeat 1 (HR1) region profile. / Spike glycoprotein S2 superfamily, coronavirus / Spike glycoprotein S2, coronavirus / Coronavirus spike glycoprotein S2 / Coronavirus spike glycoprotein S1, C-terminal / Coronavirus spike glycoprotein S1, C-terminal
Similarity search - Domain/homology
Biological speciesSevere acute respiratory syndrome coronavirus 2
synthetic construct (others)
Homo sapiens (human)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.21 Å
AuthorsLiu, H. / Wilson, I.A.
Funding support United States, 1items
OrganizationGrant numberCountry
Bill & Melinda Gates FoundationINV-004923 United States
CitationJournal: Proc Natl Acad Sci U S A / Year: 2023
Title: Fully synthetic platform to rapidly generate tetravalent bispecific nanobody-based immunoglobulins.
Authors: Laetitia Misson Mindrebo / Hejun Liu / Gabriel Ozorowski / Quoc Tran / Jordan Woehl / Irene Khalek / Jessica M Smith / Shawn Barman / Fangzhu Zhao / Celina Keating / Oliver Limbo / Megan ...Authors: Laetitia Misson Mindrebo / Hejun Liu / Gabriel Ozorowski / Quoc Tran / Jordan Woehl / Irene Khalek / Jessica M Smith / Shawn Barman / Fangzhu Zhao / Celina Keating / Oliver Limbo / Megan Verma / Jingjia Liu / Robyn L Stanfield / Xueyong Zhu / Hannah L Turner / Devin Sok / Po-Ssu Huang / Dennis R Burton / Andrew B Ward / Ian A Wilson / Joseph G Jardine /
Abstract: Nanobodies bind a target antigen with a kinetic profile similar to a conventional antibody, but exist as a single heavy chain domain that can be readily multimerized to engage antigen via multiple ...Nanobodies bind a target antigen with a kinetic profile similar to a conventional antibody, but exist as a single heavy chain domain that can be readily multimerized to engage antigen via multiple interactions. Presently, most nanobodies are produced by immunizing camelids; however, platforms for animal-free production are growing in popularity. Here, we describe the development of a fully synthetic nanobody library based on an engineered human V3-23 variable gene and a multispecific antibody-like format designed for biparatopic target engagement. To validate our library, we selected nanobodies against the SARS-CoV-2 receptor-binding domain and employed an on-yeast epitope binning strategy to rapidly map the specificities of the selected nanobodies. We then generated antibody-like molecules by replacing the V and V domains of a conventional antibody with two different nanobodies, designed as a molecular clamp to engage the receptor-binding domain biparatopically. The resulting bispecific tetra-nanobody immunoglobulins neutralized diverse SARS-CoV-2 variants with potencies similar to antibodies isolated from convalescent donors. Subsequent biochemical analyses confirmed the accuracy of the on-yeast epitope binning and structures of both individual nanobodies, and a tetra-nanobody immunoglobulin revealed that the intended mode of interaction had been achieved. This overall workflow is applicable to nearly any protein target and provides a blueprint for a modular workflow for the development of multispecific molecules.
History
DepositionSep 26, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jul 5, 2023Provider: repository / Type: Initial release
Revision 1.1Oct 25, 2023Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond / pdbx_initial_refinement_model

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Spike protein S1
B: Nanobody Nb-C4-255
H: CC12.1 Fab heavy chain
L: CC12.1 Fab light chain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)85,6985
Polymers85,4774
Non-polymers2211
Water1,18966
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)78.543, 140.387, 142.095
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number20
Space group name H-MC2221

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Components

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Antibody , 3 types, 3 molecules BHL

#2: Antibody Nanobody Nb-C4-255


Mass: 15551.948 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) synthetic construct (others) / Production host: Homo sapiens (human)
#3: Antibody CC12.1 Fab heavy chain


Mass: 23178.928 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Mus musculus (house mouse)
#4: Antibody CC12.1 Fab light chain


Mass: 23641.365 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Mus musculus (house mouse)

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Protein / Sugars / Non-polymers , 3 types, 68 molecules A

#1: Protein Spike protein S1


Mass: 23104.867 Da / Num. of mol.: 1 / Fragment: Receptor binding domain, UNP residues 333-530
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Severe acute respiratory syndrome coronavirus 2
Production host: Trichoplusia ni (cabbage looper) / References: UniProt: P0DTC2
#5: Sugar ChemComp-NAG / 2-acetamido-2-deoxy-beta-D-glucopyranose / N-acetyl-beta-D-glucosamine / 2-acetamido-2-deoxy-beta-D-glucose / 2-acetamido-2-deoxy-D-glucose / 2-acetamido-2-deoxy-glucose / N-ACETYL-D-GLUCOSAMINE


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C8H15NO6
IdentifierTypeProgram
DGlcpNAcbCONDENSED IUPAC CARBOHYDRATE SYMBOLGMML 1.0
N-acetyl-b-D-glucopyranosamineCOMMON NAMEGMML 1.0
b-D-GlcpNAcIUPAC CARBOHYDRATE SYMBOLPDB-CARE 1.0
GlcNAcSNFG CARBOHYDRATE SYMBOLGMML 1.0
#6: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 66 / Source method: isolated from a natural source / Formula: H2O

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Details

Has ligand of interestN

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 2.29 Å3/Da / Density % sol: 46.32 %
Crystal growTemperature: 293 K / Method: vapor diffusion, sitting drop / pH: 4.6
Details: 0.2 M ammonium sulfate, 0.1 M sodium acetate pH 4.6, 25% (w/v) polyethylene glycol 4000

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Data collection

DiffractionMean temperature: 110 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: APS / Beamline: 23-ID-B / Wavelength: 1.03317 Å
DetectorType: DECTRIS EIGER X 16M / Detector: PIXEL / Date: Nov 20, 2021
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.03317 Å / Relative weight: 1
ReflectionResolution: 2.2→50 Å / Num. obs: 38288 / % possible obs: 96.4 % / Redundancy: 6.5 % / Biso Wilson estimate: 32.56 Å2 / Rmerge(I) obs: 0.131 / Rpim(I) all: 0.054 / Rrim(I) all: 0.142 / Χ2: 0.866 / Net I/σ(I): 5.7 / Num. measured all: 248854
Reflection shell

Diffraction-ID: 1

Resolution (Å)Redundancy (%)Rmerge(I) obsNum. unique obsCC1/2Rpim(I) allRrim(I) allΧ2% possible all
2.2-2.246.31.04918840.6810.431.1370.72196.1
2.24-2.286.60.93919320.7440.3781.0150.72198.3
2.28-2.326.70.8719120.7720.3490.9390.7597.9
2.32-2.376.70.86919240.7520.3490.9390.75798.1
2.37-2.426.80.78819490.7850.3150.850.77199.6
2.42-2.486.80.64319210.8560.2590.6950.79798.7
2.48-2.546.70.55819490.8670.2280.6040.82498.6
2.54-2.616.50.50119530.8950.2080.5440.83998.8
2.61-2.696.40.40519580.9280.1710.440.88799.5
2.69-2.775.90.33918870.9240.1480.3710.91496.3
2.77-2.876.40.29418560.9470.1230.320.97194.3
2.87-2.9970.23119550.9680.0910.2490.99699
2.99-3.1270.18719470.980.0750.2021.0598
3.12-3.296.80.1519370.9840.0610.1631.07597.9
3.29-3.496.40.11919130.9850.050.131.09796.2
3.49-3.766.10.09418900.9890.0410.1021.05494.8
3.76-4.145.40.07417330.9910.0340.0811.02586.9
4.14-4.746.20.05518070.9930.0240.060.79589.5
4.74-5.976.60.05219400.9960.0210.0560.7195.4
5.97-506.40.04720410.9960.020.0510.61295.6

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Processing

Software
NameVersionClassification
PHENIX1.19.2_4158refinement
HKL-2000data scaling
PDB_EXTRACT3.27data extraction
HKL-2000data reduction
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 7KN5, 6XC3

7kn5

6xc3


Resolution: 2.21→49.93 Å / SU ML: 0.29 / Cross valid method: THROUGHOUT / σ(F): 1.35 / Phase error: 29.99 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.2849 1550 4.94 %
Rwork0.2344 29803 -
obs0.2368 31353 78.64 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso max: 97.53 Å2 / Biso mean: 38.4557 Å2 / Biso min: 16.01 Å2
Refinement stepCycle: final / Resolution: 2.21→49.93 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms5751 0 14 66 5831
Biso mean--53.32 33.52 -
Num. residues----753
LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Rfactor Rfree error: 0 / Total num. of bins used: 11

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkNum. reflection all% reflection obs (%)
2.21-2.280.363780.30461177125535
2.28-2.360.3367790.27891541162045
2.36-2.450.4034850.28851950203557
2.45-2.570.35221110.28142473258472
2.57-2.70.30991510.27863007315888
2.7-2.870.33151930.27723139333292
2.87-3.090.3052020.26873339354198
3.09-3.40.30771620.25263363352597
3.4-3.890.30591910.23083266345795
3.9-4.910.22751150.18633091320687
4.91-49.930.22391830.19993457364096
Refinement TLS params.Method: refined / Origin x: 8.1476 Å / Origin y: 37.7605 Å / Origin z: 0.3745 Å
111213212223313233
T0.113 Å20.0047 Å2-0.0007 Å2-0.1837 Å20.02 Å2--0.207 Å2
L0.1244 °20.1573 °2-0.1695 °2-0.757 °2-0.5214 °2--0.9021 °2
S0.0158 Å °0.0357 Å °0.0023 Å °-0.0683 Å °0.0285 Å °0.0766 Å °0.0665 Å °-0.0387 Å °-0.0463 Å °
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection detailsAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1allA334 - 527
2X-RAY DIFFRACTION1allA628
3X-RAY DIFFRACTION1allB1 - 112
4X-RAY DIFFRACTION1allH1 - 214
5X-RAY DIFFRACTION1allL1 - 213
6X-RAY DIFFRACTION1allW1 - 66

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