Journal: Cell Rep / Year: 2024 Title: Structural and functional insight into the interaction of Clostridioides difficile toxin B and FZD. Authors: Julia Kinsolving / Julien Bous / Pawel Kozielewicz / Sara Košenina / Rawan Shekhani / Lukas Grätz / Geoffrey Masuyer / Yuankai Wang / Pål Stenmark / Min Dong / Gunnar Schulte / Abstract: The G protein-coupled receptors of the Frizzled (FZD) family, in particular FZD, are receptors that are exploited by Clostridioides difficile toxin B (TcdB), the major virulence factor responsible ...The G protein-coupled receptors of the Frizzled (FZD) family, in particular FZD, are receptors that are exploited by Clostridioides difficile toxin B (TcdB), the major virulence factor responsible for pathogenesis associated with Clostridioides difficile infection. We employ a live-cell assay examining the affinity between full-length FZDs and TcdB. Moreover, we present cryoelectron microscopy structures of TcdB alone and in complex with full-length FZD, which reveal that large structural rearrangements of the combined repetitive polypeptide domain are required for interaction with FZDs and other TcdB receptors, constituting a first step for receptor recognition. Furthermore, we show that bezlotoxumab, an FDA-approved monoclonal antibody to treat Clostridioides difficile infection, favors the apo-TcdB structure and thus disrupts binding with FZD. The dynamic transition between the two conformations of TcdB also governs the stability of the pore-forming region. Thus, our work provides structural and functional insight into how conformational dynamics of TcdB determine receptor binding.
Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K
-
Electron microscopy imaging
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
Microscopy
Model: FEI TITAN KRIOS
Electron gun
Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lens
Mode: BRIGHT FIELD / Calibrated magnification: 105000 X / Nominal defocus max: 2000 nm / Nominal defocus min: 600 nm / Cs: 2.7 mm
Specimen holder
Cryogen: NITROGEN
Image recording
Average exposure time: 3 sec. / Electron dose: 50.453 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 17269 Details: Images collected in super-resolution mode, faster acquisition mode, with 4 exposures per hole
EM imaging optics
Energyfilter slit width: 20 eV
-
Processing
EM software
ID
Name
Version
Category
1
Topaz
0.2.5a
particleselection
2
EPU
2.14.0
imageacquisition
4
cryoSPARC
4.2.1
CTFcorrection
7
Coot
0.9
modelfitting
9
cryoSPARC
4.2.1
initialEulerassignment
10
cryoSPARC
4.2.1
finalEulerassignment
11
cryoSPARC
4.2.1
classification
12
cryoSPARC
4.2.1
3Dreconstruction
25
Rosetta
modelrefinement
26
PHENIX
modelrefinement
CTF correction
Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selection
Num. of particles selected: 5240176
3D reconstruction
Resolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 41523 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT
Atomic model building
Protocol: RIGID BODY FIT / Space: REAL
Atomic model building
Source name: AlphaFold / Type: in silico model
Refine LS restraints
Refine-ID
Type
Dev ideal
Number
ELECTRONMICROSCOPY
f_bond_d
0.003
19379
ELECTRONMICROSCOPY
f_angle_d
0.516
26235
ELECTRONMICROSCOPY
f_dihedral_angle_d
10.638
7126
ELECTRONMICROSCOPY
f_chiral_restr
0.045
2905
ELECTRONMICROSCOPY
f_plane_restr
0.003
3413
+
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