6XW7

Crystal structure of murine norovirus P domain in complex with Nanobody NB-5829 and glycochenodeoxycholate (GCDCA)


Experimental Data Snapshot

  • Method: X-RAY DIFFRACTION
  • Resolution: 2.15 Å
  • R-Value Free: 0.215 
  • R-Value Work: 0.171 
  • R-Value Observed: 0.174 

Starting Model: experimental
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Literature

Nanobody-Mediated Neutralization Reveals an Achilles Heel for Norovirus.

Koromyslova, A.D.Devant, J.M.Kilic, T.Sabin, C.D.Malak, V.Hansman, G.S.

(2020) J Virol 94

  • DOI: https://doi.org/10.1128/JVI.00660-20
  • Primary Citation of Related Structures:  
    6XW4, 6XW5, 6XW6, 6XW7

  • PubMed 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 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 2+ or Ca 2+ 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. IMPORTANCE 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.


  • Organizational Affiliation

    Schaller Research Group at the University of Heidelberg and DKFZ, Heidelberg, Germany anna.koromyslova@gmail.com g.hansman@dkfz.de.


Macromolecules
Find similar proteins by:  (by identity cutoff)  |  3D Structure
Entity ID: 1
MoleculeChains Sequence LengthOrganismDetailsImage
Capsid proteinA,
B,
C [auth E],
D [auth F]
309Murine norovirus 1Mutation(s): 0 
UniProt
Find proteins for Q80J94 (Norovirus (isolate Mouse/NoV/United States/MNV1/2002/GV))
Explore Q80J94 
Go to UniProtKB:  Q80J94
Entity Groups  
Sequence Clusters30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
UniProt GroupQ80J94
Sequence Annotations
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  • Reference Sequence
Find similar proteins by:  (by identity cutoff)  |  3D Structure
Entity ID: 2
MoleculeChains Sequence LengthOrganismDetailsImage
Nanobody NB-5829E [auth C],
F [auth D],
G,
H
123Vicugna pacosMutation(s): 0 
Entity Groups  
Sequence Clusters30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
Sequence Annotations
Expand
  • Reference Sequence
Small Molecules
Ligands 3 Unique
IDChains Name / Formula / InChI Key2D Diagram3D Interactions
CHO (Subject of Investigation/LOI)
Query on CHO

Download Ideal Coordinates CCD File 
K [auth A],
L [auth B],
P [auth E],
Q [auth F]
GLYCOCHENODEOXYCHOLIC ACID
C26 H43 N O5
GHCZAUBVMUEKKP-GYPHWSFCSA-N
EDO
Query on EDO

Download Ideal Coordinates CCD File 
I [auth A]
J [auth A]
N [auth E]
O [auth E]
R [auth C]
1,2-ETHANEDIOL
C2 H6 O2
LYCAIKOWRPUZTN-UHFFFAOYSA-N
MG
Query on MG

Download Ideal Coordinates CCD File 
M [auth B]MAGNESIUM ION
Mg
JLVVSXFLKOJNIY-UHFFFAOYSA-N
Experimental Data & Validation

Experimental Data

  • Method: X-RAY DIFFRACTION
  • Resolution: 2.15 Å
  • R-Value Free: 0.215 
  • R-Value Work: 0.171 
  • R-Value Observed: 0.174 
  • Space Group: C 1 2 1
Unit Cell:
Length ( Å )Angle ( ˚ )
a = 190.73α = 90
b = 124.93β = 125.3
c = 113.13γ = 90
Software Package:
Software NamePurpose
XSCALEdata scaling
REFMACrefinement
PDB_EXTRACTdata extraction
PHASERphasing
XDSdata reduction

Structure Validation

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Ligand Structure Quality Assessment 

Created with Raphaël 2.3.0Worse 01 BetterLigand structure goodness of fit to experimental dataBest fitted CHOClick on this verticalbar to view details

Entry History & Funding Information

Deposition Data


Funding OrganizationLocationGrant Number
German Federal Ministry for Education and ResearchGermanyNATION, 03VP00912

Revision History  (Full details and data files)

  • Version 1.0: 2020-04-22
    Type: Initial release
  • Version 1.1: 2020-05-06
    Changes: Database references
  • Version 1.2: 2020-07-01
    Changes: Database references
  • Version 1.3: 2024-01-24
    Changes: Data collection, Database references, Derived calculations, Refinement description
  • Version 1.4: 2024-10-23
    Changes: Structure summary