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- PDB-6xw7: Crystal structure of murine norovirus P domain in complex with Na... -

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Basic information

Entry
Database: PDB / ID: 6xw7
TitleCrystal structure of murine norovirus P domain in complex with Nanobody NB-5829 and glycochenodeoxycholate (GCDCA)
Components
  • Capsid protein
  • Nanobody NB-5829
KeywordsVIRAL PROTEIN / MNV / neutralizing nanobody / VHH / norovirus
Function / homology
Function and homology information


virion component / host cell cytoplasm
Similarity search - Function
Nucleoplasmin-like/VP (viral coat and capsid proteins) / Positive stranded ssRNA viruses / 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 ...Nucleoplasmin-like/VP (viral coat and capsid proteins) / Positive stranded ssRNA viruses / 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: 2.15 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: Data collection / Database references ...Data collection / Database references / Derived calculations / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_initial_refinement_model / pdbx_struct_conn_angle / struct_conn
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _pdbx_struct_conn_angle.ptnr1_auth_seq_id / _pdbx_struct_conn_angle.ptnr3_auth_seq_id / _pdbx_struct_conn_angle.value / _struct_conn.pdbx_dist_value / _struct_conn.ptnr2_auth_seq_id
Revision 1.4Oct 23, 2024Group: Structure summary / Category: pdbx_entry_details / pdbx_modification_feature / Item: _pdbx_entry_details.has_protein_modification

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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
E: Capsid protein
F: Capsid protein
C: Nanobody NB-5829
D: Nanobody NB-5829
G: Nanobody NB-5829
H: Nanobody NB-5829
hetero molecules


Theoretical massNumber of molelcules
Total (without water)190,84721
Polymers188,5278
Non-polymers2,31913
Water16,195899
1
A: Capsid protein
B: Capsid protein
C: Nanobody NB-5829
D: Nanobody NB-5829
hetero molecules


Theoretical massNumber of molelcules
Total (without water)95,43611
Polymers94,2644
Non-polymers1,1727
Water724
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area11150 Å2
ΔGint-12 kcal/mol
Surface area30740 Å2
MethodPISA
2
E: Capsid protein
F: Capsid protein
G: Nanobody NB-5829
H: Nanobody NB-5829
hetero molecules


Theoretical massNumber of molelcules
Total (without water)95,41110
Polymers94,2644
Non-polymers1,1486
Water724
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area10860 Å2
ΔGint-2 kcal/mol
Surface area30140 Å2
MethodPISA
Unit cell
Length a, b, c (Å)190.730, 124.930, 113.130
Angle α, β, γ (deg.)90.000, 125.300, 90.000
Int Tables number5
Space group name H-MC121
Components on special symmetry positions
IDModelComponents
11A-836-

HOH

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Components

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Protein / Antibody , 2 types, 8 molecules ABEFCDGH

#1: Protein
Capsid protein


Mass: 33683.980 Da / Num. of mol.: 4
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-5829


Mass: 13447.881 Da / Num. of mol.: 4
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

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Non-polymers , 4 types, 912 molecules

#3: Chemical
ChemComp-EDO / 1,2-ETHANEDIOL / ETHYLENE GLYCOL


Mass: 62.068 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: C2H6O2
#4: Chemical
ChemComp-CHO / GLYCOCHENODEOXYCHOLIC ACID


Mass: 449.623 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C26H43NO5 / Feature type: SUBJECT OF INVESTIGATION / Comment: detergent*YM
#5: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg
#6: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 899 / 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: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 2.92 Å3/Da / Density % sol: 57.84 %
Crystal growTemperature: 291 K / Method: vapor diffusion, hanging drop
Details: 0.2 M Sodium chloride, 0.1M Phosphate-citrate pH 4.2, 20% PEG8000

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: ESRF / Beamline: ID30B / Wavelength: 0.97856 Å
DetectorType: DECTRIS PILATUS3 6M / Detector: PIXEL / Date: May 12, 2018
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.97856 Å / Relative weight: 1
ReflectionResolution: 2.15→49.21 Å / Num. obs: 114707 / % possible obs: 96.9 % / Redundancy: 6.886 % / Biso Wilson estimate: 38.879 Å2 / CC1/2: 0.998 / Rmerge(I) obs: 0.096 / Rrim(I) all: 0.104 / Χ2: 0.959 / Net I/σ(I): 14.36 / Num. measured all: 789862 / Scaling rejects: 36
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
2.15-2.26.5360.7322.3951530870778840.8080.79590.5
2.2-2.266.9410.6412.8757622854183020.8520.69397.2
2.26-2.336.9270.5593.355600826680270.8950.60497.1
2.33-2.46.8680.4753.8553581802378020.9190.51497.2
2.4-2.486.9680.4054.4952957782076000.9410.43797.2
2.48-2.576.7090.335.4148690747572570.9530.35897.1
2.57-2.667.0260.2796.5649819731470910.9690.30197
2.66-2.776.9730.228.2347608701168270.980.23797.4
2.77-2.897.2260.17810.2247112670665200.9870.19197.2
2.89-3.047.1370.13812.7844734640862680.9920.14997.8
3.04-3.26.9590.10616.3441833613560110.9940.11498
3.2-3.396.8120.08319.9838279578056190.9960.0997.2
3.39-3.636.8190.06624.536380543453350.9980.07198.2
3.63-3.927.1810.05529.8535969509850090.9980.05998.3
3.92-4.297.0020.04235.4431934465845610.9990.04697.9
4.29-4.86.550.03837.9926952425341150.9990.04196.8
4.8-5.546.7060.03937.924517373936560.9990.04297.8
5.54-6.796.9190.0437.1621567319031170.9990.04497.7
6.79-9.66.3830.03641.2215032247923550.9990.0495
9.6-49.216.030.03447.798146138613510.9990.03797.5

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Processing

Software
NameVersionClassification
XSCALEdata scaling
REFMAC5.8.0238refinement
PDB_EXTRACT3.25data extraction
PHASERphasing
XDSdata reduction
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 3LQ6

3lq6


Resolution: 2.15→49.21 Å / Cor.coef. Fo:Fc: 0.962 / Cor.coef. Fo:Fc free: 0.943 / SU B: 5.049 / SU ML: 0.127 / Cross valid method: THROUGHOUT / σ(F): 0 / ESU R: 0.193 / ESU R Free: 0.169
Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS U VALUES : REFINED INDIVIDUALLY
RfactorNum. reflection% reflectionSelection details
Rfree0.2147 5759 5 %RANDOM
Rwork0.1714 ---
obs0.1736 108949 97.14 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å
Displacement parametersBiso max: 146.38 Å2 / Biso mean: 33.609 Å2 / Biso min: 12.07 Å2
Baniso -1Baniso -2Baniso -3
1--0.19 Å20 Å2-1.17 Å2
2--2.76 Å20 Å2
3----0.46 Å2
Refinement stepCycle: final / Resolution: 2.15→49.21 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms12858 0 161 899 13918
Biso mean--32.2 34.3 -
Num. residues----1677
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0090.01313364
X-RAY DIFFRACTIONr_bond_other_d0.0010.01712165
X-RAY DIFFRACTIONr_angle_refined_deg1.5961.64818235
X-RAY DIFFRACTIONr_angle_other_deg1.2721.56528192
X-RAY DIFFRACTIONr_dihedral_angle_1_deg8.15451670
X-RAY DIFFRACTIONr_dihedral_angle_2_deg30.54821.452668
X-RAY DIFFRACTIONr_dihedral_angle_3_deg13.95151978
X-RAY DIFFRACTIONr_dihedral_angle_4_deg19.341592
X-RAY DIFFRACTIONr_chiral_restr0.0650.21734
X-RAY DIFFRACTIONr_gen_planes_refined0.0070.0215056
X-RAY DIFFRACTIONr_gen_planes_other0.0010.022924
LS refinement shellResolution: 2.15→2.202 Å / Rfactor Rfree error: 0
RfactorNum. reflection% reflection
Rfree0.294 421 -
Rwork0.254 7451 -
obs--90.65 %

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