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- PDB-7ne1: Structure of the complex between Netrin-1 and its receptor Neogenin -

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Basic information

Entry
Database: PDB / ID: 7ne1
TitleStructure of the complex between Netrin-1 and its receptor Neogenin
Components
  • Neogenin
  • Netrin-1
KeywordsSIGNALING PROTEIN / cell surface receptor signaling / axon guidance / migration / cancer / growth cone / receptor clustering / Netrin / Neogenin / Repulsive Guidance Molecule
Function / homology
Function and homology information


regulation of glial cell migration / DSCAM interactions / chemorepulsion of axon / Cdc42 protein signal transduction / anterior/posterior axon guidance / Role of second messengers in netrin-1 signaling / Netrin-1 signaling / Regulation of commissural axon pathfinding by SLIT and ROBO / motor neuron migration / negative regulation of axon extension ...regulation of glial cell migration / DSCAM interactions / chemorepulsion of axon / Cdc42 protein signal transduction / anterior/posterior axon guidance / Role of second messengers in netrin-1 signaling / Netrin-1 signaling / Regulation of commissural axon pathfinding by SLIT and ROBO / motor neuron migration / negative regulation of axon extension / Netrin mediated repulsion signals / substrate-dependent cell migration, cell extension / mammary gland duct morphogenesis / DCC mediated attractive signaling / inner ear morphogenesis / nuclear migration / positive regulation of cell motility / regulation of synapse assembly / basement membrane / positive regulation of axon extension / glial cell proliferation / positive regulation of glial cell proliferation / cell-cell adhesion / actin cytoskeleton / Ras protein signal transduction / DNA-binding transcription factor activity, RNA polymerase II-specific / RNA polymerase II cis-regulatory region sequence-specific DNA binding / apoptotic process / regulation of transcription by RNA polymerase II / glutamatergic synapse / extracellular region / nucleoplasm / membrane / cytosol
Similarity search - Function
Neogenin, C-terminal / Neogenin C-terminus / Laminin, N-terminal / : / Laminin N-terminal (Domain VI) / Laminin N-terminal domain profile. / Laminin N-terminal domain (domain VI) / Laminin-type EGF-like (LE) domain profile. / Laminin-type EGF-like (LE) domain signature. / Laminin-type epidermal growth factor-like domai ...Neogenin, C-terminal / Neogenin C-terminus / Laminin, N-terminal / : / Laminin N-terminal (Domain VI) / Laminin N-terminal domain profile. / Laminin N-terminal domain (domain VI) / 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 / Immunoglobulin domain / Immunoglobulin I-set / Immunoglobulin I-set domain / Fibronectin type III domain / EGF-like domain signature 1. / Fibronectin type 3 domain / Immunoglobulin subtype 2 / Immunoglobulin C-2 Type / Fibronectin type-III domain profile. / Galactose-binding-like domain superfamily / Fibronectin type III / Fibronectin type III superfamily / Immunoglobulin subtype / Immunoglobulin / Ig-like domain profile. / Immunoglobulin-like domain / Immunoglobulin-like domain superfamily / Immunoglobulin-like fold / Immunoglobulins / Immunoglobulin-like / Sandwich / Mainly Beta
Similarity search - Domain/homology
sucrose octasulfate / NITRATE ION / Netrin-1 / Neogenin
Similarity search - Component
Biological speciesHomo sapiens (human)
Mus musculus (house mouse)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 3.15 Å
AuthorsRobinson, R.A. / Griffiths, S.C. / van de Haar, L.L. / Malinauskas, T. / van Battum, E.Y. / Zelina, P. / Schwab, R.A. / Karia, D. / Malinauskaite, L. / Brignani, S. ...Robinson, R.A. / Griffiths, S.C. / van de Haar, L.L. / Malinauskas, T. / van Battum, E.Y. / Zelina, P. / Schwab, R.A. / Karia, D. / Malinauskaite, L. / Brignani, S. / van den Munkhof, M. / Dudukcu, O. / De Ruiter, A.A. / Van den Heuvel, D.M.A. / Bishop, B. / Elegheert, J. / Aricescu, A.R. / Pasterkamp, R.J. / Siebold, C.
Funding support United Kingdom, 4items
OrganizationGrant numberCountry
Cancer Research UKC20724/A14414 United Kingdom
Cancer Research UKC20724/A26752 United Kingdom
European Research Council (ERC)647278 United Kingdom
Medical Research Council (MRC, United Kingdom)MR/L017776/1 United Kingdom
CitationJournal: Cell / Year: 2021
Title: Simultaneous binding of Guidance Cues NET1 and RGM blocks extracellular NEO1 signaling.
Authors: Ross A Robinson / Samuel C Griffiths / Lieke L van de Haar / Tomas Malinauskas / Eljo Y van Battum / Pavol Zelina / Rebekka A Schwab / Dimple Karia / Lina Malinauskaite / Sara Brignani / ...Authors: Ross A Robinson / Samuel C Griffiths / Lieke L van de Haar / Tomas Malinauskas / Eljo Y van Battum / Pavol Zelina / Rebekka A Schwab / Dimple Karia / Lina Malinauskaite / Sara Brignani / Marleen H van den Munkhof / Özge Düdükcü / Anna A De Ruiter / Dianne M A Van den Heuvel / Benjamin Bishop / Jonathan Elegheert / A Radu Aricescu / R Jeroen Pasterkamp / Christian Siebold /
Abstract: During cell migration or differentiation, cell surface receptors are simultaneously exposed to different ligands. However, it is often unclear how these extracellular signals are integrated. Neogenin ...During cell migration or differentiation, cell surface receptors are simultaneously exposed to different ligands. However, it is often unclear how these extracellular signals are integrated. Neogenin (NEO1) acts as an attractive guidance receptor when the Netrin-1 (NET1) ligand binds, but it mediates repulsion via repulsive guidance molecule (RGM) ligands. Here, we show that signal integration occurs through the formation of a ternary NEO1-NET1-RGM complex, which triggers reciprocal silencing of downstream signaling. Our NEO1-NET1-RGM structures reveal a "trimer-of-trimers" super-assembly, which exists in the cell membrane. Super-assembly formation results in inhibition of RGMA-NEO1-mediated growth cone collapse and RGMA- or NET1-NEO1-mediated neuron migration, by preventing formation of signaling-compatible RGM-NEO1 complexes and NET1-induced NEO1 ectodomain clustering. These results illustrate how simultaneous binding of ligands with opposing functions, to a single receptor, does not lead to competition for binding, but to formation of a super-complex that diminishes their functional outputs.
History
DepositionFeb 2, 2021Deposition site: PDBE / Processing site: PDBE
Revision 1.0Mar 31, 2021Provider: repository / Type: Initial release
Revision 1.1Apr 28, 2021Group: Database references / Category: citation
Item: _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first
Revision 1.2Jan 31, 2024Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_entity_branch_descriptor / pdbx_initial_refinement_model
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession
Revision 1.3Oct 23, 2024Group: Structure summary / Category: pdbx_entry_details / pdbx_modification_feature / Item: _pdbx_entry_details.has_protein_modification

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Netrin-1
B: Neogenin
hetero molecules


Theoretical massNumber of molelcules
Total (without water)90,96311
Polymers88,8692
Non-polymers2,0949
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: SAXS, Confirmed from Rg calculations of SAXS experiments with inline gel filtration. Also confirmed by sedimentation velocity AUC experiments and verified using interface mutants as negative controls.
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area3210 Å2
ΔGint-10 kcal/mol
Surface area41320 Å2
Unit cell
Length a, b, c (Å)150.950, 49.600, 157.250
Angle α, β, γ (deg.)90.000, 99.380, 90.000
Int Tables number5
Space group name H-MC121

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Components

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Protein , 2 types, 2 molecules AB

#1: Protein Netrin-1 / Epididymis tissue protein Li 131P


Mass: 49600.820 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: Human Netrin-1 expressed in HEK293T cells using the pHLSEC vector for secreted proteins. Contains C-terminal Rho-1D4 tag.
Source: (gene. exp.) Homo sapiens (human) / Gene: NTN1, NTN1L / Plasmid: pHLsec / Cell line (production host): HEK293T / Production host: Homo sapiens (human) / References: UniProt: O95631
#2: Protein Neogenin


Mass: 39268.199 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: Mouse Neogenin FN domain 4-6 (isoform 2 - NP_001036217.1) expressed in HEK293T cells using the pHLSEC vector for secreted proteins. Contains C-terminal His6-tag
Source: (gene. exp.) Mus musculus (house mouse) / Gene: Neo1 / Plasmid: pHLsec / Cell line (production host): HEK293T / Production host: Homo sapiens (human) / References: UniProt: Q7TQG5

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Sugars , 2 types, 5 molecules

#3: Polysaccharide 1,3,4,6-tetra-O-sulfo-beta-D-fructofuranose-(2-1)-2,3,4,6-tetra-O-sulfonato-alpha-D-glucopyranose


Type: oligosaccharide, Oligosaccharide / Class: Substrate analog / Mass: 982.803 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: oligosaccharide with reducing-end-to-reducing-end glycosidic bond
References: sucrose octasulfate
DescriptorTypeProgram
WURCS=2.0/2,2,1/[ha122h-2b_2-5_1*OSO/3=O/3=O_3*OSO/3=O/3=O_4*OSO/3=O/3=O_6*OSO/3=O/3=O][a2122h-1a_1-5_2*OSO/3=O/3=O_3*OSO/3=O/3=O_4*OSO/3=O/3=O_6*OSO/3=O/3=O]/1-2/a2-b1WURCSPDB2Glycan 1.1.0
[][b-D-Fruf1SO33SO34SO36SO3]{[(2+1)][a-D-Glcp2SO33SO34SO36SO3]{}}LINUCSPDB-CARE
#4: 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.: 4 / Source method: obtained synthetically / Formula: C8H15NO6
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

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Non-polymers , 2 types, 4 molecules

#5: Chemical ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Ca
#6: Chemical ChemComp-NO3 / NITRATE ION


Mass: 62.005 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: NO3

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Details

Has ligand of interestN
Has protein modificationY

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 3.27 Å3/Da / Density % sol: 62.36 %
Crystal growTemperature: 298.15 K / Method: vapor diffusion, sitting drop
Details: 0.2 M ammonium nitrate, 20% w/v PEG 3350, 40 mM potassium/sodium tartrate

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: Diamond / Beamline: I03 / Wavelength: 0.9763 Å
DetectorType: DECTRIS PILATUS 6M / Detector: PIXEL / Date: Mar 20, 2013
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9763 Å / Relative weight: 1
ReflectionResolution: 3.15→77.57 Å / Num. obs: 19817 / % possible obs: 97.5 % / Redundancy: 3.3 % / CC1/2: 0.993 / Rmerge(I) obs: 0.119 / Rpim(I) all: 0.118 / Net I/σ(I): 6.6
Reflection shellResolution: 3.15→3.23 Å / Mean I/σ(I) obs: 1 / Num. unique obs: 1376 / CC1/2: 0.336

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Processing

Software
NameVersionClassification
BUSTER2.10.3refinement
PDB_EXTRACT3.27data extraction
xia2data reduction
Aimlessdata scaling
PHASERphasing
Cootmodel building
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 4BQ6, 1X5I, 4PLM

4bq6

1x5i

4plm


Resolution: 3.15→74.47 Å / Cor.coef. Fo:Fc: 0.912 / Cor.coef. Fo:Fc free: 0.874 / Cross valid method: THROUGHOUT / σ(F): 0 / SU Rfree Blow DPI: 0.399
RfactorNum. reflection% reflectionSelection details
Rfree0.239 1015 5.12 %RANDOM
Rwork0.212 ---
obs0.214 19815 97.2 %-
Displacement parametersBiso max: 249.88 Å2 / Biso mean: 114.04 Å2 / Biso min: 12.09 Å2
Baniso -1Baniso -2Baniso -3
1-12.2632 Å20 Å213.6627 Å2
2---11.7064 Å20 Å2
3----0.5568 Å2
Refine analyzeLuzzati coordinate error obs: 0.46 Å
Refinement stepCycle: final / Resolution: 3.15→74.47 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms5654 0 124 0 5778
Biso mean--166.33 --
Num. residues----719
Refine LS restraints
Refine-IDTypeNumberRestraint functionWeightDev ideal
X-RAY DIFFRACTIONt_dihedral_angle_d2014SINUSOIDAL2
X-RAY DIFFRACTIONt_trig_c_planes
X-RAY DIFFRACTIONt_gen_planes1001HARMONIC5
X-RAY DIFFRACTIONt_it5944HARMONIC20
X-RAY DIFFRACTIONt_nbd0SEMIHARMONIC5
X-RAY DIFFRACTIONt_improper_torsion
X-RAY DIFFRACTIONt_pseud_angle
X-RAY DIFFRACTIONt_chiral_improper_torsion789SEMIHARMONIC5
X-RAY DIFFRACTIONt_sum_occupancies
X-RAY DIFFRACTIONt_utility_distance
X-RAY DIFFRACTIONt_utility_angle
X-RAY DIFFRACTIONt_utility_torsion
X-RAY DIFFRACTIONt_ideal_dist_contact6069SEMIHARMONIC4
X-RAY DIFFRACTIONt_bond_d5944HARMONIC20.007
X-RAY DIFFRACTIONt_angle_deg8110HARMONIC20.89
X-RAY DIFFRACTIONt_omega_torsion1.76
X-RAY DIFFRACTIONt_other_torsion16.94
LS refinement shellResolution: 3.15→3.17 Å / Rfactor Rfree error: 0 / Total num. of bins used: 50
RfactorNum. reflection% reflection
Rfree0.3365 19 4.79 %
Rwork0.248 378 -
all0.2521 397 -
obs--91.53 %
Refinement TLS params.

Method: refined / Refine-ID: X-RAY DIFFRACTION

IDL11 (°2)L12 (°2)L13 (°2)L22 (°2)L23 (°2)L33 (°2)S11 (Å °)S12 (Å °)S13 (Å °)S21 (Å °)S22 (Å °)S23 (Å °)S31 (Å °)S32 (Å °)S33 (Å °)T11 (Å2)T12 (Å2)T13 (Å2)T22 (Å2)T23 (Å2)T33 (Å2)Origin x (Å)Origin y (Å)Origin z (Å)
11.74470.14960.49591.4794-1.20423.8488-0.25780.1053-0.0865-0.21370.19590.07960.1458-0.1080.0619-0.171-0.01390.0247-0.2733-0.07750.0485-141.238-100.77972.2688
23.09260.13262.16990.21540.10433.051-0.0573-0.44240.18390.44670.059-0.2016-0.12820.167-0.00170.14070.1257-0.2051-0.0672-0.0765-0.0218-107.5745-103.213117.7954
35.3883-1.8914-0.40073.5814-0.28643.7456-0.0788-0.20350.3288-0.0001-0.02690.234-0.2368-0.26540.1057-0.34140.14750.0317-0.0496-0.1430.1248-155.3131-92.842689.8715
43.3159-0.60450.14521.1296-0.15161.06370.03350.12130.2268-0.15920.0026-0.2144-0.06480.0805-0.036-0.028-0.2995-0.12740.3611-0.262-0.281-149.6459-114.111135.8438
52.5805-0.8371-1.86121.49620.37186.17390.05390.4176-0.2103-0.30090.0194-0.27650.34090.2694-0.07340.3002-0.3449-0.1605-0.1202-0.1114-0.2794-161.2013-99.1983175.522
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection detailsAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1{ A|38 - A|293 }A38 - 293
2X-RAY DIFFRACTION2{ A|294 - A|451 }A294 - 451
3X-RAY DIFFRACTION3{ B|765 - B|864 }B765 - 864
4X-RAY DIFFRACTION4{ B|885 - B|983 }B885 - 983
5X-RAY DIFFRACTION5{ B|984 - B|1073 }B984 - 1073

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