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- PDB-6xw5: Crystal structure of murine norovirus P domain in complex with Na... -

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Basic information

Entry
Database: PDB / ID: 6xw5
TitleCrystal structure of murine norovirus P domain in complex with Nanobody NB-5820
Components
  • Capsid proteinCapsid
  • Nanobody NB-5820
KeywordsVIRAL PROTEIN / MNV / neutralizing nanobody / VHH / norovirus
Function / homology
Function and homology information


Positive stranded ssRNA viruses / Nucleoplasmin-like/VP (viral coat and capsid proteins) / Positive stranded ssRNA viruses / Calicivirus coat protein C-terminal / Calicivirus coat protein C-terminal / Calicivirus coat protein / Calicivirus coat protein / Elongation Factor Tu (Ef-tu); domain 3 / Picornavirus/Calicivirus coat protein / Viral coat protein subunit ...Positive stranded ssRNA viruses / Nucleoplasmin-like/VP (viral coat and capsid proteins) / Positive stranded ssRNA viruses / Calicivirus coat protein C-terminal / Calicivirus coat protein C-terminal / Calicivirus coat protein / Calicivirus coat protein / Elongation Factor Tu (Ef-tu); domain 3 / Picornavirus/Calicivirus coat protein / Viral coat protein subunit / Beta Barrel / Mainly Beta
Similarity search - Domain/homology
Biological speciesMurine norovirus 1
Vicugna pacos (alpaca)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.72 Å
AuthorsKilic, T. / Sabin, C. / Hansman, G.
Funding support Germany, 1items
OrganizationGrant numberCountry
German Federal Ministry for Education and ResearchNATION, 03VP00912 Germany
CitationJournal: J Virol / Year: 2020
Title: Nanobody-Mediated Neutralization Reveals an Achilles Heel for Norovirus.
Authors: Anna D Koromyslova / Jessica M Devant / Turgay Kilic / Charles D Sabin / Virginie Malak / Grant S Hansman /
Abstract: Human norovirus frequently causes outbreaks of acute gastroenteritis. Although discovered more than five decades ago, antiviral development has, until recently, been hampered by the lack of a ...Human norovirus frequently causes outbreaks of acute gastroenteritis. Although discovered more than five decades ago, antiviral development has, until recently, been hampered by the lack of a reliable human norovirus cell culture system. Nevertheless, a lot of pathogenesis studies were accomplished using murine norovirus (MNV), which can be grown routinely in cell culture. In this study, we analyzed a sizeable library of nanobodies that were raised against the murine norovirus virion with the main purpose of developing nanobody-based inhibitors. We discovered two types of neutralizing nanobodies and analyzed the inhibition mechanisms using X-ray crystallography, cryo-electron microscopy (cryo-EM), and cell culture techniques. The first type bound on the top region of the protruding (P) domain. Interestingly, this nanobody binding region closely overlapped the MNV receptor-binding site and collectively shared numerous P domain-binding residues. In addition, we showed that these nanobodies competed with the soluble receptor, and this action blocked virion attachment to cultured cells. The second type bound at a dimeric interface on the lower side of the P dimer. We discovered that these nanobodies disrupted a structural change in the capsid associated with binding cofactors (i.e., metal cations/bile acid). Indeed, we found that capsids underwent major conformational changes following addition of Mg or Ca Ultimately, these nanobodies directly obstructed a structural modification reserved for a postreceptor attachment stage. Altogether, our new data show that nanobody-based inhibition could occur by blocking functional and structural capsid properties. This research discovered and analyzed two different types of MNV-neutralizing nanobodies. The top-binding nanobodies sterically inhibited the receptor-binding site, whereas the dimeric-binding nanobodies interfered with a structural modification associated with cofactor binding. Moreover, we found that the capsid contained a number of vulnerable regions that were essential for viral replication. In fact, the capsid appeared to be organized in a state of flux, which could be important for cofactor/receptor-binding functions. Blocking these capsid-binding events with nanobodies directly inhibited essential capsid functions. Moreover, a number of MNV-specific nanobody binding epitopes were comparable to human norovirus-specific nanobody inhibitors. Therefore, this additional structural and inhibition information could be further exploited in the development of human norovirus antivirals.
History
DepositionJan 23, 2020Deposition site: PDBE / Processing site: PDBE
Revision 1.0Apr 22, 2020Provider: repository / Type: Initial release
Revision 1.1May 6, 2020Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year
Revision 1.2Jul 1, 2020Group: Database references / Category: citation / Item: _citation.journal_volume / _citation.title
Revision 1.3Jan 24, 2024Group: Advisory / Data collection ...Advisory / Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_initial_refinement_model / pdbx_unobs_or_zero_occ_atoms
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Capsid protein
B: Capsid protein
C: Nanobody NB-5820
D: Nanobody NB-5820
hetero molecules


Theoretical massNumber of molelcules
Total (without water)94,06314
Polymers93,3834
Non-polymers68110
Water12,827712
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: isothermal titration calorimetry
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area10700 Å2
ΔGint10 kcal/mol
Surface area31470 Å2
MethodPISA
Unit cell
Length a, b, c (Å)101.720, 101.720, 228.520
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number92
Space group name H-MP41212
Components on special symmetry positions
IDModelComponents
11A-847-

HOH

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Components

#1: Protein Capsid protein / Capsid


Mass: 33320.570 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Murine norovirus 1 / Production host: Escherichia coli BL21 (bacteria) / References: UniProt: Q80J94
#2: Antibody Nanobody NB-5820


Mass: 13370.796 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Vicugna pacos (alpaca) / Plasmid: pHEN6C / Production host: Escherichia coli (E. coli) / Strain (production host): WK6
#3: Chemical
ChemComp-EDO / 1,2-ETHANEDIOL / ETHYLENE GLYCOL / Ethylene glycol


Mass: 62.068 Da / Num. of mol.: 9 / Source method: obtained synthetically / Formula: C2H6O2
#4: Chemical ChemComp-TRS / 2-AMINO-2-HYDROXYMETHYL-PROPANE-1,3-DIOL / TRIS BUFFER / Tris


Mass: 122.143 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C4H12NO3 / Comment: pH buffer*YM
#5: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 712 / Source method: isolated from a natural source / Formula: H2O
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: 3.17 Å3/Da / Density % sol: 61.14 %
Crystal growTemperature: 291 K / Method: vapor diffusion, hanging drop / Details: 25% PEG3000, 0.1M Tris pH 8.5

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: ESRF / Beamline: ID29 / Wavelength: 1.07227 Å
DetectorType: DECTRIS PILATUS 6M / Detector: PIXEL / Date: Jul 19, 2018
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.07227 Å / Relative weight: 1
ReflectionResolution: 1.72→49.86 Å / Num. obs: 127876 / % possible obs: 99.9 % / Redundancy: 13.019 % / Biso Wilson estimate: 30.811 Å2 / CC1/2: 1 / Rmerge(I) obs: 0.066 / Rrim(I) all: 0.069 / Χ2: 1.092 / Net I/σ(I): 22.34 / Num. measured all: 1664812 / Scaling rejects: 29
Reflection shell

Diffraction-ID: 1

Resolution (Å)Redundancy (%)Rmerge(I) obsMean I/σ(I) obsNum. measured obsNum. possibleNum. unique obsCC1/2Rrim(I) all% possible all
1.72-1.7612.4570.7913.04115194934392470.9350.82499
1.76-1.8112.4490.574.13113158909090900.9640.595100
1.81-1.8613.7030.4565.58121750888688850.9830.474100
1.86-1.9213.6230.3646.89117208860686040.9860.378100
1.92-1.9813.4340.2828.86111983833683360.990.293100
1.98-2.0513.1710.22510.97106682810081000.9920.234100
2.05-2.1312.3930.18413.0796813781278120.9940.192100
2.13-2.2213.3290.15116.53100596754775470.9960.158100
2.22-2.3213.6740.13219.0699136725072500.9970.137100
2.32-2.4313.5310.11322.0793704692569250.9970.118100
2.43-2.5613.360.09825.3887885657865780.9980.102100
2.56-2.7212.4230.0829.5377865626962680.9980.083100
2.72-2.913.240.06735.7978446592559250.9990.07100
2.9-3.1413.4470.05742.2973840549154910.9990.059100
3.14-3.4412.9960.04848.1366438511251120.9980.05100
3.44-3.8411.9520.04352.5155148461446140.9990.044100
3.84-4.4412.1280.03657.7950211414041400.9990.038100
4.44-5.4312.8960.03461.19454193522352210.035100
5.43-7.6811.9170.03257.18332142788278710.034100
7.68-49.8612.2470.02662.72201221652164310.02799.5

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Processing

Software
NameVersionClassification
REFMAC5.8.0238refinement
PDB_EXTRACT3.22data extraction
XDSdata reduction
XSCALEdata scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 3LQ6

3lq6


Resolution: 1.72→49.86 Å / Cor.coef. Fo:Fc: 0.971 / Cor.coef. Fo:Fc free: 0.962 / SU B: 1.784 / SU ML: 0.057 / Cross valid method: THROUGHOUT / σ(F): 0 / ESU R: 0.08 / ESU R Free: 0.082
Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS U VALUES : REFINED INDIVIDUALLY
RfactorNum. reflection% reflectionSelection details
Rfree0.1911 6299 4.9 %RANDOM
Rwork0.1614 ---
obs0.1629 121577 99.86 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å
Displacement parametersBiso max: 109.51 Å2 / Biso mean: 27.631 Å2 / Biso min: 15.3 Å2
Baniso -1Baniso -2Baniso -3
1-1.08 Å20 Å20 Å2
2--1.08 Å20 Å2
3----2.17 Å2
Refinement stepCycle: final / Resolution: 1.72→49.86 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms6484 0 44 712 7240
Biso mean--36.32 37.97 -
Num. residues----848
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0120.0136795
X-RAY DIFFRACTIONr_bond_other_d0.0010.0176165
X-RAY DIFFRACTIONr_angle_refined_deg1.7351.6499266
X-RAY DIFFRACTIONr_angle_other_deg1.4641.56714279
X-RAY DIFFRACTIONr_dihedral_angle_1_deg7.615868
X-RAY DIFFRACTIONr_dihedral_angle_2_deg31.58621.525341
X-RAY DIFFRACTIONr_dihedral_angle_3_deg12.074151020
X-RAY DIFFRACTIONr_dihedral_angle_4_deg20.1361548
X-RAY DIFFRACTIONr_chiral_restr0.080.2877
X-RAY DIFFRACTIONr_gen_planes_refined0.0110.027757
X-RAY DIFFRACTIONr_gen_planes_other0.0010.021499
LS refinement shellResolution: 1.72→1.763 Å / Rfactor Rfree error: 0
RfactorNum. reflection% reflection
Rfree0.272 447 -
Rwork0.249 8781 -
obs--98.92 %

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