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- PDB-9h4r: Structure of fertilization-blocking monoclonal antibody IE-3 VHVL... -

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Basic information

Entry
Database: PDB / ID: 9h4r
TitleStructure of fertilization-blocking monoclonal antibody IE-3 VHVL bound to the ZP-N1 domain of mouse ZP2 (crystal form I)
Components
  • Heavy chain variable (VH) domain of anti-ZP2 monoclonal antibody IE-3
  • Light chain variable (VL) domain of anti-ZP2 monoclonal antibody IE-3
  • Zona pellucida sperm-binding protein 2
KeywordsCELL ADHESION / Zona pellucida / ZP2 / ZP-N domain / ZP-N1 domain / ZP module / ZP domain / egg-sperm recognition / gamete interaction / immunocontraception / monoclonal antibody / IE-3 / IE3 / heavy chain variable domain / light chain variable domain
Function / homology
Function and homology information


Interaction With Cumulus Cells And The Zona Pellucida / acrosin binding / structural constituent of egg coat / egg coat / prevention of polyspermy / binding of sperm to zona pellucida / multivesicular body / : / endoplasmic reticulum / extracellular region ...Interaction With Cumulus Cells And The Zona Pellucida / acrosin binding / structural constituent of egg coat / egg coat / prevention of polyspermy / binding of sperm to zona pellucida / multivesicular body / : / endoplasmic reticulum / extracellular region / identical protein binding / plasma membrane
Similarity search - Function
Zona pellucida sperm-binding protein 2, Ig-like domain / Zona pellucida sperm-binding protein 2, Ig-like domain / Zona pellucida sperm-binding protein 2, Ig-like domain / : / : / : / : / ZP-N domain / Zona pellucida domain, conserved site / ZP domain signature. ...Zona pellucida sperm-binding protein 2, Ig-like domain / Zona pellucida sperm-binding protein 2, Ig-like domain / Zona pellucida sperm-binding protein 2, Ig-like domain / : / : / : / : / ZP-N domain / Zona pellucida domain, conserved site / ZP domain signature. / Zona pellucida, ZP-C domain / ZP-C domain / Zona pellucida (ZP) domain / ZP domain profile. / Zona pellucida domain
Similarity search - Domain/homology
Zona pellucida sperm-binding protein 2
Similarity search - Component
Biological speciesMus musculus (house mouse)
Rattus norvegicus (Norway rat)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.53 Å
AuthorsDioguardi, E. / De Sanctis, D. / Jovine, L.
Funding support Sweden, European Union, 6items
OrganizationGrant numberCountry
Swedish Research Council2012-5093 Sweden
Swedish Research Council2016-03999 Sweden
Swedish Research Council2020-04936 Sweden
European Research Council (ERC)ERC 260759European Union
Knut and Alice Wallenberg Foundation2018.0042 Sweden
European Molecular Biology Organization (EMBO)EMBO Young InvestigatorEuropean Union
Citation
Journal: Proc.Natl.Acad.Sci.USA / Year: 2025
Title: Structural basis of ZP2-targeted female nonhormonal contraception.
Authors: Dioguardi, E. / Stsiapanava, A. / Fahrenkamp, E. / Han, L. / de Sanctis, D. / Inzunza, J. / Jovine, L.
#1: Journal: Proc Natl Acad Sci U S A / Year: 1980
Title: Synthesis of zona pellucida proteins by denuded and follicle-enclosed mouse oocytes during culture in vitro.
Authors: Bleil, J.D. / Wassarman, P.M.
#2: Journal: Dev Biol / Year: 1981
Title: Mammalian sperm-egg interaction: fertilization of mouse eggs triggers modification of the major zona pellucida glycoprotein, ZP2.
Authors: Bleil, J.D. / Beall, C.F. / Wassarman, P.M.
#3: Journal: Cell / Year: 1982
Title: Biosynthesis of the major zona pellucida glycoprotein secreted by oocytes during mammalian oogenesis.
Authors: Greve, J.M. / Salzmann, G.S. / Roller, R.J. / Wassarman, P.M.
#4: Journal: Mol Cell Biol / Year: 1990
Title: Oocyte-specific expression of mouse Zp-2: developmental regulation of the zona pellucida genes.
Authors: Liang, L.F. / Chamow, S.M. / Dean, J.
#5: Journal: Nature / Year: 2008
Title: Crystal structure of the ZP-N domain of ZP3 reveals the core fold of animal egg coats.
Authors: Monne, M. / Han, L. / Schwend, T. / Burendahl, S. / Jovine, L.
#6: Journal: J Cell Biol / Year: 2014
Title: A single domain of the ZP2 zona pellucida protein mediates gamete recognition in mice and humans.
Authors: Avella, M.A. / Baibakov, B. / Dean, J.
#7: Journal: Dev Biol / Year: 1985
Title: Monoclonal antibodies to the murine zona pellucida protein with sperm receptor activity: effects on fertilization and early development.
Authors: East, I.J. / Gulyas, B.J. / Dean, J.
#8: Journal: Dev Biol / Year: 1984
Title: Monoclonal antibodies to the major protein of the murine zona pellucida: effects on fertilization and early development.
Authors: East, I.J. / Mattison, D.R. / Dean, J.
#9: Journal: Curr Biol / Year: 2015
Title: Vectored antibody gene delivery mediates long-term contraception.
Authors: Li, J. / Olvera, A.I. / Akbari, O.S. / Moradian, A. / Sweredoski, M.J. / Hess, S. / Hay, B.A.
#10: Journal: Biol Reprod / Year: 1999
Title: A contraceptive peptide vaccine targeting sulfated glycoprotein ZP2 of the mouse zona pellucida.
Authors: Sun, W. / Lou, Y.H. / Dean, J. / Tung, K.S.
#11: Journal: Cell / Year: 2017
Title: Structural Basis of Egg Coat-Sperm Recognition at Fertilization.
Authors: Raj, I. / Sadat Al Hosseini, H. / Dioguardi, E. / Nishimura, K. / Han, L. / Villa, A. / de Sanctis, D. / Jovine, L.
#12: Journal: Cell / Year: 2024
Title: ZP2 cleavage blocks polyspermy by modulating the architecture of the egg coat.
Authors: Shunsuke Nishio / Chihiro Emori / Benjamin Wiseman / Dirk Fahrenkamp / Elisa Dioguardi / Sara Zamora-Caballero / Marcel Bokhove / Ling Han / Alena Stsiapanava / Blanca Algarra / Yonggang Lu ...Authors: Shunsuke Nishio / Chihiro Emori / Benjamin Wiseman / Dirk Fahrenkamp / Elisa Dioguardi / Sara Zamora-Caballero / Marcel Bokhove / Ling Han / Alena Stsiapanava / Blanca Algarra / Yonggang Lu / Mayo Kodani / Rachel E Bainbridge / Kayla M Komondor / Anne E Carlson / Michael Landreh / Daniele de Sanctis / Shigeki Yasumasu / Masahito Ikawa / Luca Jovine /
Abstract: Following the fertilization of an egg by a single sperm, the egg coat or zona pellucida (ZP) hardens and polyspermy is irreversibly blocked. These events are associated with the cleavage of the N- ...Following the fertilization of an egg by a single sperm, the egg coat or zona pellucida (ZP) hardens and polyspermy is irreversibly blocked. These events are associated with the cleavage of the N-terminal region (NTR) of glycoprotein ZP2, a major subunit of ZP filaments. ZP2 processing is thought to inactivate sperm binding to the ZP, but its molecular consequences and connection with ZP hardening are unknown. Biochemical and structural studies show that cleavage of ZP2 triggers its oligomerization. Moreover, the structure of a native vertebrate egg coat filament, combined with AlphaFold predictions of human ZP polymers, reveals that two protofilaments consisting of type I (ZP3) and type II (ZP1/ZP2/ZP4) components interlock into a left-handed double helix from which the NTRs of type II subunits protrude. Together, these data suggest that oligomerization of cleaved ZP2 NTRs extensively cross-links ZP filaments, rigidifying the egg coat and making it physically impenetrable to sperm.
History
DepositionOct 21, 2024Deposition site: PDBE / Processing site: PDBE
Revision 1.0Mar 12, 2025Provider: repository / Type: Initial release
Revision 1.1Apr 23, 2025Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _citation_author.name

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Zona pellucida sperm-binding protein 2
H: Heavy chain variable (VH) domain of anti-ZP2 monoclonal antibody IE-3
L: Light chain variable (VL) domain of anti-ZP2 monoclonal antibody IE-3
B: Zona pellucida sperm-binding protein 2
X: Heavy chain variable (VH) domain of anti-ZP2 monoclonal antibody IE-3
Y: Light chain variable (VL) domain of anti-ZP2 monoclonal antibody IE-3


Theoretical massNumber of molelcules
Total (without water)78,2656
Polymers78,2656
Non-polymers00
Water11,097616
1
A: Zona pellucida sperm-binding protein 2
H: Heavy chain variable (VH) domain of anti-ZP2 monoclonal antibody IE-3
L: Light chain variable (VL) domain of anti-ZP2 monoclonal antibody IE-3


Theoretical massNumber of molelcules
Total (without water)39,1323
Polymers39,1323
Non-polymers00
Water543
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
2
B: Zona pellucida sperm-binding protein 2
X: Heavy chain variable (VH) domain of anti-ZP2 monoclonal antibody IE-3
Y: Light chain variable (VL) domain of anti-ZP2 monoclonal antibody IE-3


Theoretical massNumber of molelcules
Total (without water)39,1323
Polymers39,1323
Non-polymers00
Water543
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)91.640, 92.290, 91.170
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number19
Space group name H-MP212121
Space group name HallP2ac2ab
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
d_1ens_1(chain "A" and resid 43 through 136)
d_2ens_1(chain "B" and resid 43 through 136)
d_1ens_2(chain "H" and (resid 21 through 77 or resid 79 through 139))
d_2ens_2(chain "X" and (resid 21 through 77 or resid 79 through 139))
d_1ens_3(chain "L" and (resid 20 through 104 or resid 106 through 132))
d_2ens_3(chain "Y" and (resid 20 through 104 or resid 106 through 132))

NCS domain segments:
Dom-IDComponent-IDEns-IDBeg auth comp-IDBeg label comp-IDEnd auth comp-IDEnd label comp-IDAuth asym-IDLabel asym-IDAuth seq-IDLabel seq-ID
d_11ens_1PROPROALAALAAA43 - 1369 - 102
d_21ens_1PROPROALAALABD43 - 1369 - 102
d_11ens_2VALVALTHRTHRHB21 - 775 - 61
d_12ens_2TYRTYRSERSERHB79 - 13963 - 123
d_21ens_2VALVALTHRTHRXE21 - 775 - 61
d_22ens_2TYRTYRSERSERXE79 - 13963 - 123
d_11ens_3GLYGLYVALVALLC20 - 1043 - 87
d_12ens_3ALAALAILEILELC106 - 13289 - 115
d_21ens_3GLYGLYVALVALYF20 - 1043 - 87
d_22ens_3ALAALAILEILEYF106 - 13289 - 115

NCS ensembles :
ID
ens_1
ens_2
ens_3

NCS oper:
IDCodeMatrixVector
1given(0.0579281295329, 0.998204685536, -0.015222929538), (0.998315318377, -0.0578707508401, 0.00418345453508), (0.0032949815563, -0.0154396234445, -0.999875372796)24.3773488139, -25.7542446444, -23.2804162453
2given(0.0575332342217, 0.998168928155, -0.0186739879546), (0.998330782289, -0.0574276772852, 0.00614092963136), (0.00505728139413, -0.0189961245459, -0.999806766909)24.180273522, -25.2722697468, -23.8215741507
3given(0.0582936555392, 0.998106465817, -0.0196298909609), (0.998058905133, -0.0586999976916, -0.020802215183), (-0.0219151000315, -0.018379150314, -0.999590883924)24.3725811888, -25.5932943159, -23.8334162281

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Components

#1: Protein Zona pellucida sperm-binding protein 2 / Zona pellucida glycoprotein 2 / Zp-2 / Zona pellucida protein A


Mass: 12994.641 Da / Num. of mol.: 2 / Fragment: UNP residues 35-138 / Mutation: N83S
Source method: isolated from a genetically manipulated source
Details: DBREF 9H4R A 35 138 UNP P20239 ZP2_MOUSE 35 138 SEQADV 9H4R SER A 83 UNP P20239 ASN 83 ENGINEERED MUTATION SEQADV 9H4R LEU A 139 UNP P20239 EXPRESSION TAG SEQADV 9H4R GLU A 140 UNP P20239 ...Details: DBREF 9H4R A 35 138 UNP P20239 ZP2_MOUSE 35 138 SEQADV 9H4R SER A 83 UNP P20239 ASN 83 ENGINEERED MUTATION SEQADV 9H4R LEU A 139 UNP P20239 EXPRESSION TAG SEQADV 9H4R GLU A 140 UNP P20239 EXPRESSION TAG SEQADV 9H4R HIS A 141 UNP P20239 EXPRESSION TAG SEQADV 9H4R HIS A 142 UNP P20239 EXPRESSION TAG SEQADV 9H4R HIS A 143 UNP P20239 EXPRESSION TAG SEQADV 9H4R HIS A 144 UNP P20239 EXPRESSION TAG SEQADV 9H4R HIS A 145 UNP P20239 EXPRESSION TAG SEQADV 9H4R HIS A 146 UNP P20239 EXPRESSION TAG
Source: (gene. exp.) Mus musculus (house mouse) / Gene: Zp2, Zp-2, Zpa / Plasmid: pHLsec / Cell line (production host): HEK293T / Production host: Homo sapiens (human) / References: UniProt: P20239
#2: Antibody Heavy chain variable (VH) domain of anti-ZP2 monoclonal antibody IE-3


Mass: 13290.527 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Details: The cross-reference for 9H4R chains H and X are residues 20-139 of GenBank entry QCC30352 (https://www.ncbi.nlm.nih.gov/protein/QCC30352), with the preceding 3 residues (17-ETG-19) being an expression tag.
Source: (gene. exp.) Rattus norvegicus (Norway rat) / Cell line: Hybridoma / Plasmid: pHLsec3 / Cell line (production host): HEK293T / Production host: Homo sapiens (human)
#3: Antibody Light chain variable (VL) domain of anti-ZP2 monoclonal antibody IE-3


Mass: 12847.207 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Details: The cross-reference for 9H4R chains L and Y are residues 21-134 of GenBank entry QCC30353 (https://www.ncbi.nlm.nih.gov/protein/QCC30353), with the preceding 3 residues (18-ETG-20) being an expression tag.
Source: (gene. exp.) Rattus norvegicus (Norway rat) / Cell line: Hybridoma / Plasmid: pHLsec3 / Cell line (production host): HEK293T / Production host: Homo sapiens (human)
#4: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 616 / Source method: isolated from a natural source / Formula: H2O
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: 1.99 Å3/Da / Density % sol: 38.09 %
Crystal growTemperature: 293 K / Method: vapor diffusion, hanging drop / pH: 6.5
Details: 24% (v/v) PEG 3350, 0.2 M sodium tartrate, 0.1 M Na-HEPES pH 6.5

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: ESRF / Beamline: ID29 / Wavelength: 0.976251 Å
DetectorType: DECTRIS PILATUS 6M-F / Detector: PIXEL / Date: Sep 10, 2014
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.976251 Å / Relative weight: 1
Reflection twinOperator: k,h,-l / Fraction: 0.3
ReflectionResolution: 1.53→29.164 Å / Num. obs: 115130 / % possible obs: 98.5 % / Redundancy: 3.3 % / Biso Wilson estimate: 37.27 Å2 / CC1/2: 1 / Rpim(I) all: 0.016 / Rrim(I) all: 0.03 / Net I/σ(I): 18.6
Reflection shellResolution: 1.53→1.58 Å / Redundancy: 3.4 % / Mean I/σ(I) obs: 1 / Num. unique obs: 11474 / CC1/2: 0.55 / Rpim(I) all: 0.721 / Rrim(I) all: 1.376 / % possible all: 99.2

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Processing

Software
NameVersionClassification
PHENIX1.21.2_5419+SVNrefinement
MxCuBEdata collection
XDSdata reduction
XSCALEdata scaling
PHASERphasing
PHENIXmodel building
Cootmodel building
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: P43-Q138 of PDB ID 5II6; chain H of PDB ID 4AMK; chain L of PDB ID 3MBX

5ii6

4amk

3mbx


Resolution: 1.53→29.16 Å / Cross valid method: FREE R-VALUE / σ(F): 1.5 / Phase error: 35.8263
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflection
Rfree0.2329 6384 5.85 %
Rwork0.2058 102781 -
obs0.2095 109165 93.38 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.1 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 36.88 Å2
Refinement stepCycle: LAST / Resolution: 1.53→29.16 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms5152 0 0 616 5768
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00255268
X-RAY DIFFRACTIONf_angle_d0.57647146
X-RAY DIFFRACTIONf_chiral_restr0.0446784
X-RAY DIFFRACTIONf_plane_restr0.0035918
X-RAY DIFFRACTIONf_dihedral_angle_d11.64351882
Refine LS restraints NCS
Ens-IDDom-IDAsym-IDAuth asym-IDRefine-IDTypeRms dev position (Å)
ens_1d_2AAX-RAY DIFFRACTIONTorsion NCS0.440181632686
ens_2d_2BHX-RAY DIFFRACTIONTorsion NCS0.402672708597
ens_3d_2CLX-RAY DIFFRACTIONTorsion NCS0.631927043366
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
1.53-1.560.3612580.36274504X-RAY DIFFRACTION78
1.56-1.580.34612630.33924661X-RAY DIFFRACTION80.5
1.58-1.620.35032790.34374658X-RAY DIFFRACTION80.91
1.62-1.650.32942790.33964764X-RAY DIFFRACTION81.81
1.65-1.680.32222850.33134858X-RAY DIFFRACTION84.34
1.68-1.720.35232910.32415035X-RAY DIFFRACTION86.65
1.72-1.770.29612900.31455052X-RAY DIFFRACTION87.27
1.77-1.810.30062920.30585082X-RAY DIFFRACTION88.24
1.81-1.870.27183060.2975115X-RAY DIFFRACTION87.51
1.87-1.930.2853080.28385252X-RAY DIFFRACTION90.63
1.93-20.31213020.28355368X-RAY DIFFRACTION92.19
2-2.080.25823280.27085362X-RAY DIFFRACTION92.15
2.08-2.170.30993230.26225409X-RAY DIFFRACTION92.65
2.17-2.290.26743230.26035407X-RAY DIFFRACTION92.66
2.29-2.430.26292990.24135422X-RAY DIFFRACTION92.68
2.43-2.620.28693130.24345370X-RAY DIFFRACTION91.51
2.62-2.880.25533380.21865378X-RAY DIFFRACTION91.29
2.88-3.290.2032810.19565473X-RAY DIFFRACTION92.59
3.29-4.150.19272870.15785428X-RAY DIFFRACTION90.95
4.15-29.160.18393710.13885551X-RAY DIFFRACTION89.73

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