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- PDB-8qw4: FZD3 in complex with nanobody 9 -

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Basic information

Entry
Database: PDB / ID: 8qw4
TitleFZD3 in complex with nanobody 9
Components
  • Frizzled-3
  • Nanobody Nb9
KeywordsCELL ADHESION / FZD3 Nanobody
Function / homology
Function and homology information


dopaminergic neuron axon guidance / serotonergic neuron axon guidance / cell proliferation in midbrain / negative regulation of mitotic cell cycle, embryonic / establishment of planar polarity / midbrain morphogenesis / Wnt receptor activity / non-canonical Wnt signaling pathway / motor neuron migration / sympathetic ganglion development ...dopaminergic neuron axon guidance / serotonergic neuron axon guidance / cell proliferation in midbrain / negative regulation of mitotic cell cycle, embryonic / establishment of planar polarity / midbrain morphogenesis / Wnt receptor activity / non-canonical Wnt signaling pathway / motor neuron migration / sympathetic ganglion development / filopodium tip / Wnt-protein binding / post-anal tail morphogenesis / commissural neuron axon guidance / negative regulation of execution phase of apoptosis / PCP/CE pathway / Class B/2 (Secretin family receptors) / Wnt signaling pathway, planar cell polarity pathway / inner ear morphogenesis / positive regulation of neuroblast proliferation / hair follicle development / lateral plasma membrane / canonical Wnt signaling pathway / response to electrical stimulus / presynaptic active zone membrane / PDZ domain binding / Asymmetric localization of PCP proteins / neural tube closure / G protein-coupled receptor activity / neuron differentiation / Ca2+ pathway / apical plasma membrane / response to xenobiotic stimulus / axon / neuronal cell body / dendrite / glutamatergic synapse / cell surface / plasma membrane / cytoplasm
Similarity search - Function
Frizzled 3, cysteine-rich domain / Frizzled/Smoothened, transmembrane domain / Frizzled/Smoothened family membrane region / Frizzled/Smoothened family membrane region / Frizzled/secreted frizzled-related protein / Frizzled / Frizzled domain / Frizzled cysteine-rich domain superfamily / Fz domain / Frizzled (fz) domain profile. ...Frizzled 3, cysteine-rich domain / Frizzled/Smoothened, transmembrane domain / Frizzled/Smoothened family membrane region / Frizzled/Smoothened family membrane region / Frizzled/secreted frizzled-related protein / Frizzled / Frizzled domain / Frizzled cysteine-rich domain superfamily / Fz domain / Frizzled (fz) domain profile. / GPCR, family 2-like / G-protein coupled receptors family 2 profile 2.
Similarity search - Domain/homology
Biological speciesHomo sapiens (human)
Lama glama (llama)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.9 Å
AuthorsZhao, Y. / Jones, E.Y.
Funding support United Kingdom, 1items
OrganizationGrant numberCountry
Wellcome Trust223133/Z/21/Z United Kingdom
CitationJournal: Nat Commun / Year: 2024
Title: Structural insights into Frizzled3 through nanobody modulators.
Authors: James Hillier / Yuguang Zhao / Loic Carrique / Tomas Malinauskas / Reinis R Ruza / Tao-Hsin Chang / Gangshun Yi / Helen M E Duyvesteyn / Jing Yu / Weixian Lu / Els Pardon / Jan Steyaert / ...Authors: James Hillier / Yuguang Zhao / Loic Carrique / Tomas Malinauskas / Reinis R Ruza / Tao-Hsin Chang / Gangshun Yi / Helen M E Duyvesteyn / Jing Yu / Weixian Lu / Els Pardon / Jan Steyaert / Yanan Zhu / Tao Ni / E Yvonne Jones /
Abstract: The Wnt receptor Frizzled3 (FZD3) is important for brain axonal development and cancer progression. We report structures of FZD3 in complex with extracellular and intracellular binding nanobodies (Nb) ...The Wnt receptor Frizzled3 (FZD3) is important for brain axonal development and cancer progression. We report structures of FZD3 in complex with extracellular and intracellular binding nanobodies (Nb). The crystal structure of Nb8 in complex with the FZD3 cysteine-rich domain (CRD) reveals that the nanobody binds at the base of the lipid-binding groove and can compete with Wnt5a. Nb8 fused with the Dickkopf-1 C-terminal domain behaves as a FZD3-specific Wnt surrogate, activating β-catenin signalling. The cryo-EM structure of FZD3 in complex with Nb9 reveals partially resolved density for the CRD, which exhibits positional flexibility, and a transmembrane conformation that resembles active GPCRs. Nb9 binds to the cytoplasmic region of FZD3 at the putative Dishevelled (DVL) or G protein-binding site, competes with DVL binding, and inhibits GαS coupling. In combination, our FZD3 structures with nanobody modulators map extracellular and intracellular interaction surfaces of functional, and potentially therapeutic, relevance.
History
DepositionOct 18, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Sep 4, 2024Provider: repository / Type: Initial release
Revision 1.1Oct 23, 2024Group: Data collection / Structure summary
Category: em_admin / pdbx_entry_details / pdbx_modification_feature
Item: _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Frizzled-3
B: Nanobody Nb9


Theoretical massNumber of molelcules
Total (without water)74,6442
Polymers74,6442
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein Frizzled-3


Mass: 60559.270 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: FZD3 / Production host: Homo sapiens (human) / References: UniProt: Q9NPG1
#2: Antibody Nanobody Nb9


Mass: 14084.658 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Lama glama (llama) / Production host: Escherichia coli (E. coli)
Has protein modificationY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: CELL / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: FZD3 Nanobody Nb9 complex / Type: CELL / Entity ID: all / Source: MULTIPLE SOURCES
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human)
Buffer solutionpH: 8
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2000 nm / Nominal defocus min: 1000 nm
Image recordingElectron dose: 40 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

EM softwareName: PHENIX / Version: 1.18.2_3874: / Category: model refinement
CTF correctionType: NONE
3D reconstructionResolution: 2.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 330717 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0073562
ELECTRON MICROSCOPYf_angle_d1.0594826
ELECTRON MICROSCOPYf_dihedral_angle_d41.226475
ELECTRON MICROSCOPYf_chiral_restr0.058550
ELECTRON MICROSCOPYf_plane_restr0.008589

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