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Yorodumi- PDB-6xw4: Crystal structure of murine norovirus P domain in complex with Na... -
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-Basic information
Entry | Database: PDB / ID: 6xw4 | ||||||
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Title | Crystal structure of murine norovirus P domain in complex with Nanobody NB-5867 | ||||||
Components |
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Keywords | VIRAL PROTEIN / MNV / neutralizing nanobody / VHH / norovirus | ||||||
Function / homology | Function and homology information | ||||||
Biological species | Murine norovirus 1 Vicugna pacos (alpaca) | ||||||
Method | X-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.19 Å | ||||||
Authors | Kilic, T. / Sabin, C. / Hansman, G. | ||||||
Funding support | Germany, 1items
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Citation | Journal: 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 |
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-Structure visualization
Structure viewer | Molecule: MolmilJmol/JSmol |
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-Downloads & links
-Download
PDBx/mmCIF format | 6xw4.cif.gz | 185.6 KB | Display | PDBx/mmCIF format |
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PDB format | pdb6xw4.ent.gz | 143.7 KB | Display | PDB format |
PDBx/mmJSON format | 6xw4.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 6xw4_validation.pdf.gz | 461.8 KB | Display | wwPDB validaton report |
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Full document | 6xw4_full_validation.pdf.gz | 466.4 KB | Display | |
Data in XML | 6xw4_validation.xml.gz | 35.4 KB | Display | |
Data in CIF | 6xw4_validation.cif.gz | 51.3 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/xw/6xw4 ftp://data.pdbj.org/pub/pdb/validation_reports/xw/6xw4 | HTTPS FTP |
-Related structure data
Related structure data | 6xw5C 6xw6C 6xw7C 3lq6S S: Starting model for refinement C: citing same article (ref.) |
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Similar structure data |
-Links
-Assembly
Deposited unit |
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1 |
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Unit cell |
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-Components
#1: Protein | 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 | Mass: 14195.776 Da / Num. of mol.: 2 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Vicugna pacos (alpaca) / Production host: Escherichia coli (E. coli) / Variant (production host): WK6 #3: Chemical | ChemComp-EDO / #4: Water | ChemComp-HOH / | Has ligand of interest | N | |
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-Experimental details
-Experiment
Experiment | Method: X-RAY DIFFRACTION / Number of used crystals: 1 |
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-Sample preparation
Crystal | Density Matthews: 3.17 Å3/Da / Density % sol: 61.19 % |
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Crystal grow | Temperature: 291 K / Method: vapor diffusion, hanging drop Details: 1.0 M Lithium chloride 0.1 M Citric acid pH 5.0 20%(w/v) PEG 6000 |
-Data collection
Diffraction | Mean temperature: 100 K / Serial crystal experiment: N | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Diffraction source | Source: SYNCHROTRON / Site: ESRF / Beamline: ID30B / Wavelength: 0.97856 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Detector | Type: DECTRIS PILATUS3 X 6M / Detector: PIXEL / Date: May 13, 2018 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Radiation | Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Radiation wavelength | Wavelength: 0.97856 Å / Relative weight: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Reflection | Resolution: 2.19→47.38 Å / Num. obs: 62254 / % possible obs: 99.4 % / Redundancy: 6.503 % / Biso Wilson estimate: 34.419 Å2 / CC1/2: 0.996 / Rmerge(I) obs: 0.138 / Rrim(I) all: 0.15 / Χ2: 0.959 / Net I/σ(I): 10.11 / Num. measured all: 404860 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Reflection shell | Diffraction-ID: 1
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-Processing
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Refinement | Method to determine structure: MOLECULAR REPLACEMENT Starting model: 3LQ6 Resolution: 2.19→47.38 Å / Cor.coef. Fo:Fc: 0.958 / Cor.coef. Fo:Fc free: 0.933 / SU B: 5.071 / SU ML: 0.123 / Cross valid method: THROUGHOUT / σ(F): 0 / ESU R: 0.184 / ESU R Free: 0.168 / Stereochemistry target values: MAXIMUM LIKELIHOOD Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS U VALUES : REFINED INDIVIDUALLY
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Solvent computation | Ion probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: MASK | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Displacement parameters | Biso max: 121.56 Å2 / Biso mean: 30.375 Å2 / Biso min: 9.08 Å2
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Refinement step | Cycle: final / Resolution: 2.19→47.38 Å
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Refine LS restraints |
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LS refinement shell | Resolution: 2.193→2.25 Å / Rfactor Rfree error: 0 / Total num. of bins used: 20
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