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Crystal structure of the ZP-N domain of ZP3 reveals the core fold of animal egg coats

Nature volume 456, pages 653–657 (2008)Cite this article

Abstract

Species-specific recognition between the egg extracellular matrix (zona pellucida) and sperm is the first, crucial step of mammalian fertilization1. Zona pellucida filament components ZP3 and ZP2 act as sperm receptors, and mice lacking either of the corresponding genes produce oocytes without a zona pellucida and are completely infertile2. Like their counterparts in the vitelline envelope of non-mammalian eggs and many other secreted eukaryotic proteins, zona pellucida subunits polymerize using a ‘zona pellucida (ZP) domain’ module3,4,5, whose conserved amino-terminal part (ZP-N) was suggested to constitute a domain of its own6. No atomic structure has been reported for ZP domain proteins, and there is no structural information on any conserved vertebrate protein that is essential for fertilization and directly involved in egg–sperm binding. Here we describe the 2.3 ångström (Å) resolution structure of the ZP-N fragment of mouse primary sperm receptor ZP3. The ZP-N fold defines a new immunoglobulin superfamily subtype with a β-sheet extension characterized by an E′ strand and an invariant tyrosine residue implicated in polymerization. The structure strongly supports the presence of ZP-N repeats within the N-terminal region of ZP2 and other vertebrate zona pellucida/vitelline envelope proteins, with implications for overall egg coat architecture, the post-fertilization block to polyspermy and speciation. Moreover, it provides an important framework for understanding human diseases caused by mutations in ZP domain proteins and developing new methods of non-hormonal contraception.

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Figure 1: Overall structure of the ZP-N domain of ZP3.
Figure 2: Key contacts involving conserved ZP-N domain residues.
Figure 3: ZP-N domain interaction sites.
Figure 4: Model of the ZP-N domain repeat region of ZP2.

Accession codes

Primary accessions

Protein Data Bank

Data deposits

Atomic coordinates and structure factors have been deposited with the Protein Data Bank under accession codes 3D4C (crystal form I), 3D4G (crystal form II) and 3EF7 (crystal form III).

References

  1. Nixon, B., Aitken, R. J. & McLaughlin, E. A. New insights into the molecular mechanisms of sperm-egg interaction. Cell. Mol. Life Sci. 64, 1805–1823 (2007)

    CAS  PubMed  Google Scholar 

  2. Wassarman, P. M. Zona pellucida glycoproteins. J. Biol. Chem. 283, 24285–24289 (2008)

    CAS  PubMed  PubMed Central  Google Scholar 

  3. Bork, P. & Sander, C. A large domain common to sperm receptors (Zp2 and Zp3) and TGF-β type III receptor. FEBS Lett. 300, 237–240 (1992)

    CAS  PubMed  Google Scholar 

  4. Jovine, L., Qi, H., Williams, Z., Litscher, E. & Wassarman, P. M. The ZP domain is a conserved module for polymerization of extracellular proteins. Nature Cell Biol. 4, 457–461 (2002)

    CAS  PubMed  Google Scholar 

  5. Jovine, L., Darie, C. C., Litscher, E. S. & Wassarman, P. M. Zona pellucida domain proteins. Annu. Rev. Biochem. 74, 83–114 (2005)

    CAS  PubMed  Google Scholar 

  6. Jovine, L., Janssen, W. G., Litscher, E. S. & Wassarman, P. M. The PLAC1-homology region of the ZP domain is sufficient for protein polymerisation. BMC Biochem. 7, 11 (2006)

    PubMed  PubMed Central  Google Scholar 

  7. Llorca, O., Trujillo, A., Blanco, F. J. & Bernabeu, C. Structural model of human endoglin, a transmembrane receptor responsible for hereditary hemorrhagic telangiectasia. J. Mol. Biol. 365, 694–705 (2007)

    CAS  PubMed  Google Scholar 

  8. Boja, E. S., Hoodbhoy, T., Fales, H. M. & Dean, J. Structural characterization of native mouse zona pellucida proteins using mass spectrometry. J. Biol. Chem. 278, 34189–34202 (2003)

    CAS  PubMed  Google Scholar 

  9. Kanai, S. et al. Disulfide linkage patterns of pig zona pellucida glycoproteins ZP3 and ZP4. Mol. Reprod. Dev. 75, 847–856 (2008)

    CAS  PubMed  Google Scholar 

  10. Smyth, D. R., Mrozkiewicz, M. K., McGrath, W. J., Listwan, P. & Kobe, B. Crystal structures of fusion proteins with large-affinity tags. Protein Sci. 12, 1313–1322 (2003)

    CAS  PubMed  PubMed Central  Google Scholar 

  11. Halaby, D. M., Poupon, A. & Mornon, J. The immunoglobulin fold family: sequence analysis and 3D structure comparisons. Protein Eng. 12, 563–571 (1999)

    CAS  PubMed  Google Scholar 

  12. Yang, Y. et al. Structural basis for dimerization of ICAM-1 on the cell surface. Mol. Cell 14, 269–276 (2004)

    CAS  PubMed  Google Scholar 

  13. Doren, S. et al. Incorporation of mouse zona pellucida proteins into the envelope of Xenopus laevis oocytes. Dev. Genes Evol. 209, 330–339 (1999)

    CAS  PubMed  Google Scholar 

  14. ten Dijke, P., Goumans, M. J. & Pardali, E. Endoglin in angiogenesis and vascular diseases. Angiogenesis 11, 79–89 (2008)

    PubMed  Google Scholar 

  15. Legan, P. K. et al. A deafness mutation isolates a second role for the tectorial membrane in hearing. Nature Neurosci. 8, 1035–1042 (2005)

    CAS  PubMed  Google Scholar 

  16. Callebaut, I., Mornon, J. P. & Monget, P. Isolated ZP-N domains constitute the N-terminal extensions of Zona Pellucida proteins. Bioinformatics 23, 1871–1874 (2007)

    CAS  PubMed  Google Scholar 

  17. Lindsay, L. L. & Hedrick, J. L. Proteolysis of Xenopus laevis egg envelope ZPA triggers envelope hardening. Biochem. Biophys. Res. Commun. 324, 648–654 (2004)

    CAS  PubMed  Google Scholar 

  18. Tian, J., Gong, H. & Lennarz, W. J. Xenopus laevis sperm receptor gp69/64 glycoprotein is a homolog of the mammalian sperm receptor ZP2. Proc. Natl Acad. Sci. USA 96, 829–834 (1999)

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  19. Wassarman, P. M. & Mortillo, S. Structure of the mouse egg extracellular coat, the zona pellucida. Int. Rev. Cytol. 130, 85–110 (1991)

    CAS  PubMed  Google Scholar 

  20. Tsubamoto, H. et al. Expression of recombinant human zona pellucida protein 2 and its binding capacity to spermatozoa. Biol. Reprod. 61, 1649–1654 (1999)

    CAS  PubMed  Google Scholar 

  21. Hasegawa, A., Hamada, Y., Shigeta, M. & Koyama, K. Contraceptive potential of synthetic peptides of zona pellucida protein (ZPA). J. Reprod. Immunol. 53, 91–98 (2002)

    CAS  PubMed  Google Scholar 

  22. Sun, W., Lou, Y. H., Dean, J. & Tung, K. S. A contraceptive peptide vaccine targeting sulfated glycoprotein ZP2 of the mouse zona pellucida. Biol. Reprod. 60, 900–907 (1999)

    CAS  PubMed  Google Scholar 

  23. Bleil, J. D., Beall, C. F. & Wassarman, P. M. Mammalian sperm-egg interaction: fertilization of mouse eggs triggers modification of the major zona pellucida glycoprotein, ZP2. Dev. Biol. 86, 189–197 (1981)

    CAS  PubMed  Google Scholar 

  24. Rankin, T. L. et al. Fertility and taxon-specific sperm binding persist after replacement of mouse sperm receptors with human homologs. Dev. Cell 5, 33–43 (2003)

    CAS  PubMed  Google Scholar 

  25. Aricescu, A. R. & Jones, E. Y. Immunoglobulin superfamily cell adhesion molecules: zippers and signals. Curr. Opin. Cell Biol. 19, 543–550 (2007)

    CAS  PubMed  Google Scholar 

  26. Familiari, G., Heyn, R., Relucenti, M. & Sathananthan, H. Structural changes of the zona pellucida during fertilization and embryo development. Front. Biosci. 13, 6730–6751 (2008)

    PubMed  Google Scholar 

  27. Makalowski, W. & Boguski, M. S. Evolutionary parameters of the transcribed mammalian genome: an analysis of 2,820 orthologous rodent and human sequences. Proc. Natl Acad. Sci. USA 95, 9407–9412 (1998)

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  28. Swanson, W. J., Yang, Z., Wolfner, M. F. & Aquadro, C. F. Positive Darwinian selection drives the evolution of several female reproductive proteins in mammals. Proc. Natl Acad. Sci. USA 98, 2509–2514 (2001)

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  29. Turner, L. M. & Hoekstra, H. E. Adaptive evolution of fertilization proteins within a genus: variation in ZP2 and ZP3 in deer mice (Peromyscus). Mol. Biol. Evol. 23, 1656–1669 (2006)

    CAS  PubMed  Google Scholar 

  30. Galindo, B. E., Vacquier, V. D. & Swanson, W. J. Positive selection in the egg receptor for abalone sperm lysin. Proc. Natl Acad. Sci. USA 100, 4639–4643 (2003)

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  31. Center, R. J. et al. Crystallization of a trimeric human T cell leukemia virus type 1 gp21 ectodomain fragment as a chimera with maltose-binding protein. Protein Sci. 7, 1612–1619 (1998)

    CAS  PubMed  PubMed Central  Google Scholar 

  32. Kabsch, W. Automatic processing of rotation diffraction data from crystals of initially unknown symmetry and cell constants. J. Appl. Cryst. 26, 795–800 (1993)

    CAS  Google Scholar 

  33. McCoy, A. J. et al. Phaser crystallographic software. J. Appl. Cryst. 40, 658–674 (2007)

    CAS  Google Scholar 

  34. Cowtan, K. General quadratic functions in real and reciprocal space and their application to likelihood phasing. Acta Crystallogr. D 56, 1612–1621 (2000)

    CAS  PubMed  Google Scholar 

  35. Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D 60, 2126–2132 (2004)

    PubMed  Google Scholar 

  36. Davis, I. W., Arendall, W. B., Richardson, D. C. & Richardson, J. S. The backrub motion: how protein backbone shrugs when a sidechain dances. Structure 14, 265–274 (2006)

    CAS  PubMed  Google Scholar 

  37. Read, R. J. Improved Fourier coefficients for maps using phases from partial structures with errors. Acta Crystallogr. A 42, 140–149 (1986)

    Google Scholar 

  38. Brünger, A. T. Version 1.2 of the Crystallography and NMR system. Nature Protocols 2, 2728–2733 (2007)

    PubMed  Google Scholar 

  39. Somoza, J. R. et al. Holographic methods in X-ray crystallography. IV. A fast algorithm and its application to macromolecular crystallography. Acta Crystallogr. A 51, 691–708 (1995)

    PubMed  Google Scholar 

  40. Afonine, P. V., Grosse-Kunstleve, R. W. & Adams, P. D. The Phenix refinement framework. CCP4 Newsletter 42, contribution 8 (2005)

  41. Painter, J. & Merritt, E. A. Optimal description of a protein structure in terms of multiple groups undergoing TLS motion. Acta Crystallogr. D 62, 439–450 (2006)

    PubMed  Google Scholar 

  42. Murshudov, G. N., Vagin, A. A. & Dodson, E. J. Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr. D 53, 240–255 (1997)

    CAS  PubMed  Google Scholar 

  43. van den Bedem, H., Lotan, I., Latombe, J. C. & Deacon, A. M. Real-space protein-model completion: an inverse-kinematics approach. Acta Crystallogr. D 61, 2–13 (2005)

    PubMed  Google Scholar 

  44. Evans, G. & Pettifer, R. F. CHOOCH: a program for deriving anomalous-scattering factors from X-ray fluorescence spectra. J. Appl. Cryst. 34, 82–86 (2001)

    CAS  Google Scholar 

  45. Davis, I. W. et al. MolProbity: all-atom contacts and structure validation for proteins and nucleic acids. Nucleic Acids Res. 35, W375–W383 (2007)

    ADS  PubMed  PubMed Central  Google Scholar 

  46. Kleywegt, G. J. & Harris, M. R. ValLigURL: a server for ligand-structure comparison and validation. Acta Crystallogr. D 63, 935–938 (2007)

    CAS  PubMed  Google Scholar 

  47. Gille, C. & Frommel, C. STRAP: editor for STRuctural Alignments of Proteins. Bioinformatics 17, 377–378 (2001)

    CAS  PubMed  Google Scholar 

  48. O’Sullivan, O., Suhre, K., Abergel, C., Higgins, D. G. & Notredame, C. 3DCoffee: combining protein sequences and structures within multiple sequence alignments. J. Mol. Biol. 340, 385–395 (2004)

    PubMed  Google Scholar 

  49. Fiser, A. & Sali, A. Modeller: generation and refinement of homology-based protein structure models. Methods Enzymol. 374, 461–491 (2003)

    CAS  PubMed  Google Scholar 

  50. Bork, P. Consensus. <http://coot.embl.de/Alignment/consensus.html>

  51. Landau, M. et al. ConSurf 2005: the projection of evolutionary conservation scores of residues on protein structures. Nucleic Acids Res. 33, W299–W302 (2005)

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  52. Krissinel, E. & Henrick, K. Inference of macromolecular assemblies from crystalline state. J. Mol. Biol. 372, 774–797 (2007)

    CAS  PubMed  Google Scholar 

  53. Petrek, M. et al. CAVER: a new tool to explore routes from protein clefts, pockets and cavities. BMC Bioinformatics 7, 316 (2006)

    PubMed  PubMed Central  Google Scholar 

  54. Baker, N. A., Sept, D., Joseph, S., Holst, M. J. & McCammon, J. A. Electrostatics of nanosystems: application to microtubules and the ribosome. Proc. Natl Acad. Sci. USA 98, 10037–10041 (2001)

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  55. Krissinel, E. & Henrick, K. Secondary-structure matching (SSM), a new tool for fast protein structure alignment in three dimensions. Acta Crystallogr. D 60, 2256–2268 (2004)

    CAS  PubMed  Google Scholar 

  56. Holm, L. & Sander, C. Dali: a network tool for protein structure comparison. Trends Biochem. Sci. 20, 478–480 (1995)

    CAS  PubMed  Google Scholar 

  57. Liang, S., Zhang, C., Liu, S. & Zhou, Y. Protein binding site prediction using an empirical scoring function. Nucleic Acids Res. 34, 3698–3707 (2006)

    CAS  PubMed  PubMed Central  Google Scholar 

  58. Ofran, Y. & Rost, B. Protein-protein interaction hotspots carved into sequences. PLOS Comput. Biol. 3, e119 (2007)

    ADS  PubMed  PubMed Central  Google Scholar 

  59. Neuvirth, H., Raz, R. & Schreiber, G. ProMate: a structure based prediction program to identify the location of protein-protein binding sites. J. Mol. Biol. 338, 181–199 (2004)

    CAS  PubMed  Google Scholar 

  60. Porollo, A. & Meller, J. Prediction-based fingerprints of protein-protein interactions. Proteins 66, 630–645 (2007)

    CAS  PubMed  Google Scholar 

  61. Negi, S. S. & Braun, W. Statistical analysis of physical-chemical properties and prediction of protein-protein interfaces. J. Mol. Model. 13, 1157–1167 (2007)

    CAS  PubMed  PubMed Central  Google Scholar 

  62. DeLano, W. L. The PyMOL Molecular Graphics System (DeLano Scientific, 2002)

    Google Scholar 

  63. Shi, J., Blundell, T. L. & Mizuguchi, K. FUGUE: sequence-structure homology recognition using environment-specific substitution tables and structure-dependent gap penalties. J. Mol. Biol. 310, 243–257 (2001)

    CAS  PubMed  Google Scholar 

  64. Stebbings, L. A. & Mizuguchi, K. HOMSTRAD: recent developments of the Homologous Protein Structure Alignment Database. Nucleic Acids Res. 32, D203–D207 (2004)

    CAS  PubMed  PubMed Central  Google Scholar 

  65. Eddy, S. R. Profile hidden Markov models. Bioinformatics 14, 755–763 (1998)

    CAS  Google Scholar 

  66. Brooks, B. R. et al. CHARMM: a program for macromolecular energy, minimization, and dynamics calculations. J. Comput. Chem. 4, 187–217 (1983)

    CAS  Google Scholar 

  67. Bohne-Lang, A. & von der Lieth, C. W. GlyProt: in silico glycosylation of proteins. Nucleic Acids Res. 33, W214–W219 (2005)

    CAS  PubMed  PubMed Central  Google Scholar 

  68. Jorgensen, W. L., Chandrasekhar, J., Madura, J. D., Impey, R. W. & Klein, M. L. Comparison of simple potential functions for simulating liquid water. J. Chem. Phys. 79, 926–935 (1983)

    ADS  CAS  Google Scholar 

  69. Burendahl, S., Treuter, E. & Nilsson, L. Molecular dynamics simulations of human LRH-1: the impact of ligand binding in a constitutively active nuclear receptor. Biochemistry 47, 5205–5215 (2008)

    CAS  PubMed  Google Scholar 

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Acknowledgements

Acknowledgments This work was supported by Karolinska Institutet, the Swedish Research Council (grant 2005-5102) and European Community (Marie Curie ERG 31055). We thank P. Nordlund and G. Schneider for access to the Stockholm SGC robotic crystallization facility and beamtime; H. Belrhali, J. McCarthy, R. Ravelli and M. Walsh for assistance at ESRF; P. Afonine, R. Grosse-Kunstleve and P. Zwart for help with phenix.refine; G. Murshudov for help with REFMAC; C. Chothia, F. Cotelli, J.-Å. Gustafsson, R. Herbst-Irmer, A. Kohl, R. Ladenstein, M. Letarte, E. Litscher, E. Morgunova, A. Murzin, K. Nagai, L. Nilsson, D. Rhodes, R. Toftgård and P. Wassarman for discussions and comments.

Author Contributions M.M., L.H. and L.J. generated constructs and performed protein expression and purification. T.S. performed mass spectrometric analysis. S.B. carried out molecular dynamics simulations. L.J. and M.M. crystallized proteins, determined structures and wrote the manuscript.

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  1. Magnus Monné and Ling Han: These authors contributed equally to this work.

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  1. Department of Biosciences and Nutrition, Karolinska Institutet, Hälsovägen 7, SE-141 57 Huddinge, Sweden,

    Magnus Monné, Ling Han, Thomas Schwend, Sofia Burendahl & Luca Jovine

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  1. Magnus Monné
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Monné, M., Han, L., Schwend, T. et al. Crystal structure of the ZP-N domain of ZP3 reveals the core fold of animal egg coats. Nature 456, 653–657 (2008). https://doi.org/10.1038/nature07599

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