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- PDB-9az3: Cryo-EM reveals molecular mechanisms underlying the inhibitory ef... -

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Basic information

Entry
Database: PDB / ID: 9az3
TitleCryo-EM reveals molecular mechanisms underlying the inhibitory effect of netrin-4 on laminin matrix formation
Components
  • Laminin subunit gamma-1
  • Netrin-4
KeywordsSTRUCTURAL PROTEIN/INHIBITOR / Netrin-4 / Laminin G1 / Complex / Basement membrane / STRUCTURAL PROTEIN-INHIBITOR complex
Function / homology
Function and homology information


regulation of branching involved in salivary gland morphogenesis by extracellular matrix-epithelial cell signaling / Netrin-1 signaling / laminin-11 complex / laminin-1 complex / laminin-10 complex / regulation of basement membrane organization / L1CAM interactions / hemidesmosome assembly / laminin-1 binding / positive regulation of integrin-mediated signaling pathway ...regulation of branching involved in salivary gland morphogenesis by extracellular matrix-epithelial cell signaling / Netrin-1 signaling / laminin-11 complex / laminin-1 complex / laminin-10 complex / regulation of basement membrane organization / L1CAM interactions / hemidesmosome assembly / laminin-1 binding / positive regulation of integrin-mediated signaling pathway / Laminin interactions / EGR2 and SOX10-mediated initiation of Schwann cell myelination / protein complex involved in cell-matrix adhesion / Formation of the dystrophin-glycoprotein complex (DGC) / endoderm development / extracellular matrix structural constituent / MET activates PTK2 signaling / neuron remodeling / positive regulation of muscle cell differentiation / maintenance of blood-brain barrier / basement membrane / Non-integrin membrane-ECM interactions / extracellular matrix disassembly / ECM proteoglycans / Degradation of the extracellular matrix / positive regulation of cell adhesion / substrate adhesion-dependent cell spreading / positive regulation of epithelial cell proliferation / Post-translational protein phosphorylation / Regulation of Insulin-like Growth Factor (IGF) transport and uptake by Insulin-like Growth Factor Binding Proteins (IGFBPs) / cell migration / : / protein-containing complex assembly / cell adhesion / endoplasmic reticulum lumen / extracellular space / extracellular exosome / extracellular region / plasma membrane
Similarity search - Function
Netrin-4, NTR domain / Laminin IV / Laminin B (Domain IV) / Laminin IV type A domain profile. / Laminin B domain / Laminin, N-terminal / : / Laminin N-terminal (Domain VI) / Laminin N-terminal domain profile. / Laminin N-terminal domain (domain VI) ...Netrin-4, NTR domain / Laminin IV / Laminin B (Domain IV) / Laminin IV type A domain profile. / Laminin B domain / Laminin, N-terminal / : / Laminin N-terminal (Domain VI) / Laminin N-terminal domain profile. / Laminin N-terminal domain (domain VI) / : / Laminin/attractin EGF domain / Laminin-type EGF-like (LE) domain profile. / Laminin-type EGF-like (LE) domain signature. / Laminin-type epidermal growth factor-like domai / Laminin EGF domain / Laminin-type EGF domain / Netrin C-terminal Domain / Netrin module, non-TIMP type / UNC-6/NTR/C345C module / Netrin domain / NTR domain profile. / Tissue inhibitor of metalloproteinases-like, OB-fold / Epidermal growth factor-like domain. / EGF-like domain signature 1. / EGF-like domain signature 2. / EGF-like domain
Similarity search - Domain/homology
Laminin subunit gamma-1 / Netrin-4
Similarity search - Component
Biological speciesHomo sapiens (human)
Mus musculus (house mouse)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.9 Å
AuthorsKulczyk, A.W.
Funding support United States, 1items
OrganizationGrant numberCountry
Other government United States
CitationJournal: Nat Commun / Year: 2025
Title: Cryo-EM reveals molecular mechanisms underlying the inhibitory effect of netrin-4 on laminin matrix formation.
Authors: Arkadiusz W Kulczyk / Karen K McKee / Peter D Yurchenco /
Abstract: Netrin-4 is a tumor suppressor that interferes with formation of the laminin lattice. We employed cryo-electron microscopy to determine a structure of the protein complex consisting of the N-terminal ...Netrin-4 is a tumor suppressor that interferes with formation of the laminin lattice. We employed cryo-electron microscopy to determine a structure of the protein complex consisting of the N-terminal fragments from netrin-4 and laminin γ1. The structure reveals that netrin-4 binds laminin γ1 at the molecular interface where laminin β1 would have bound, thus inhibiting the assembly of the heterotrimeric laminin polymer nodes consisting of α1, β1, and γ1 subunits, and their polymerization into the extracellular lattice. The four orders of magnitude higher affinity of the netrin-4-laminin γ1 interaction results from the larger buried surface area than the one formed by β1 and γ1 laminins and greater electrostatic surface complementarity. Our findings, supported by site-directed mutagenesis, solid-phase binding analysis, laminin polymerization, and Schwann cell assays, collectively demonstrate that, in addition to inhibiting laminin polymerization, netrin-4 disassembles the pre-existing laminin lattice. The structure has the potential to facilitate the development of novel therapies for cancer treatment.
History
DepositionMar 9, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Aug 20, 2025Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
G: Laminin subunit gamma-1
N: Netrin-4
hetero molecules


Theoretical massNumber of molelcules
Total (without water)67,8799
Polymers66,3312
Non-polymers1,5487
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Laminin subunit gamma-1 / Laminin B2 chain / Laminin-1 subunit gamma / Laminin-10 subunit gamma / Laminin-11 subunit gamma / ...Laminin B2 chain / Laminin-1 subunit gamma / Laminin-10 subunit gamma / Laminin-11 subunit gamma / Laminin-2 subunit gamma / Laminin-3 subunit gamma / Laminin-4 subunit gamma / Laminin-6 subunit gamma / Laminin-7 subunit gamma / Laminin-8 subunit gamma / Laminin-9 subunit gamma / S-laminin subunit gamma / S-LAM gamma


Mass: 34152.922 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: LAMC1, LAMB2 / Production host: Homo sapiens (human) / References: UniProt: P11047
#2: Protein Netrin-4 / Beta-netrin


Mass: 32177.734 Da / Num. of mol.: 1 / Fragment: UNP residues 43-326
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Gene: Ntn4 / Production host: Homo sapiens (human) / References: UniProt: Q9JI33
#3: Sugar
ChemComp-NAG / 2-acetamido-2-deoxy-beta-D-glucopyranose / N-acetyl-beta-D-glucosamine / 2-acetamido-2-deoxy-beta-D-glucose / 2-acetamido-2-deoxy-D-glucose / 2-acetamido-2-deoxy-glucose / N-ACETYL-D-GLUCOSAMINE


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 7 / Source method: obtained synthetically / Formula: C8H15NO6 / Feature type: SUBJECT OF INVESTIGATION
IdentifierTypeProgram
DGlcpNAcbCONDENSED IUPAC CARBOHYDRATE SYMBOLGMML 1.0
N-acetyl-b-D-glucopyranosamineCOMMON NAMEGMML 1.0
b-D-GlcpNAcIUPAC CARBOHYDRATE SYMBOLPDB-CARE 1.0
GlcNAcSNFG CARBOHYDRATE SYMBOLGMML 1.0
Has ligand of interestY
Has protein modificationY

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Experimental details

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Experiment

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

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Sample preparation

ComponentName: Net4dC-LmG1dC / Type: COMPLEX
Details: A dimeric complex of the N-terminal fragments from netrin-4 and laminin gamma 1
Entity ID: #1-#2 / Source: RECOMBINANT
Molecular weightValue: 0.061 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human)
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid type: Quantifoil
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: SPOT SCAN
Electron lensMode: BRIGHT FIELD / Nominal magnification: 130000 X / Nominal defocus max: 2200 nm / Nominal defocus min: 800 nm
Image recordingElectron dose: 1.58 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

EM software
IDNameVersionCategory
9PHENIXmodel refinement
12cryoSPARC4classification
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 71000 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL

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