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Structure of IZUMO1–JUNO reveals sperm–oocyte recognition during mammalian fertilization

Nature volume 534, pages 566–569 (2016)Cite this article

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Abstract

Fertilization is a fundamental process in sexual reproduction, creating a new individual through the combination of male and female gametes1,2,3,4. The IZUMO1 sperm membrane protein5 and its counterpart oocyte receptor JUNO6 have been identified as essential factors for sperm–oocyte interaction and fusion. However, the mechanism underlying their specific recognition remains poorly defined. Here, we show the crystal structures of human IZUMO1, JUNO and the IZUMO1–JUNO complex, establishing the structural basis for the IZUMO1–JUNO-mediated sperm–oocyte interaction. IZUMO1 exhibits an elongated rod-shaped structure comprised of a helical bundle IZUMO domain and an immunoglobulin-like domain that are each firmly anchored to an intervening β-hairpin region through conserved disulfide bonds. The central β-hairpin region of IZUMO1 provides the main platform for JUNO binding, while the surface located behind the putative JUNO ligand binding pocket is involved in IZUMO1 binding. Structure-based mutagenesis analysis confirms the biological importance of the IZUMO1–JUNO interaction. This structure provides a major step towards elucidating an essential phase of fertilization and it will contribute to the development of new therapeutic interventions for fertility, such as contraceptive agents.

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Figure 1: IZUMO1–JUNO interaction.
Figure 2: Structure of IZUMO1 and JUNO.
Figure 3: Structure of the IZUMO1–JUNO complex.
Figure 4: IZUMO1 containing interface mutations abolishes cell–oocyte binding.

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Protein Data Bank

Data deposits

The coordinates and structure-factor data of human IZUMO1, JUNO (form 1 and form 2), IZUMO1-JUNO complex (form 1, form 2, and form3) have been deposited in the Protein Data Bank under the accession numbers 5JK9, 5JKA, 5JKB, 5JKC, 5JKD and 5JKE, respectively.

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Acknowledgements

We thank the beamline staff members at the Photon Factory and SPring-8 for their assistance with data collection. We thank Y. Yamada and D. Liebschner for assistance with S-SAD data collection at PF-1A. This research is supported by the Platform Project for Supporting in Drug Discovery and Life Science Research (Platform for Drug Discovery, Informatics, and Structural Life Science) from the Japan Agency for Medical Research and Development (AMED). This work was supported by a Grant-in-Aid from the Japanese Ministry of Education, Culture, Sports, Science, and Technology (U.O., S.U., N.I. and T.S.); CREST, JST (T.S.); the Takeda Science Foundation (U.O., N.I. and T.S.); the Mochida Memorial Foundation for Medical and Pharmaceutical Research (U.O.); and the Daiichi Sankyo Foundation of Life Science (U.O.).

Author information

Author notes

  1. Umeharu Ohto and Hanako Ishida: These authors contributed equally to this work.

Authors and Affiliations

  1. Graduate School of Pharmaceutical Sciences, The University of Tokyo, Hongo, Bunkyo-ku, 113-0033, Tokyo, Japan

    Umeharu Ohto, Hanako Ishida & Toshiyuki Shimizu

  2. Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, 565-0871, Japan

    Elena Krayukhina & Susumu Uchiyama

  3. Okazaki Institute for Integrative Bioscience, National Institutes of Natural Sciences, 5-1 Higashiyama, Myodaiji, Okazaki, 444-8787, Aichi, Japan

    Susumu Uchiyama

  4. Department of Cell Science, Institute of Biomedical Sciences, School of Medicine, Fukushima Medical University, 1 Hikarigaoka, Fukushima City, 960-1295, Fukushima, Japan

    Naokazu Inoue

Authors
  1. Umeharu Ohto
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Contributions

H.I. expressed and purified recombinant proteins and performed size-exclusion chromatography and isothermal titration calorimetry experiments. H.I. and U.O. performed crystallization and structure determination. E.K and S.U performed AUC analyses. N.I. performed cellular assays. U.O and T.S. directed the research and wrote the paper with assistance from all other authors.

Corresponding authors

Correspondence to Umeharu Ohto or Toshiyuki Shimizu.

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The authors declare no competing financial interests.

Additional information

Reviewer Information Nature thanks K. Melcher, M. Okabe and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Extended data figures and tables

Extended Data Figure 1 Sequence alignments of IZUMO1 and JUNO.

Sequence alignments of IZUMO1 (top) and JUNO (bottom) from Homo sapiens, Macaca mulatta, Equus caballus, Bos tauras or Bos mutus, Oryctolagus cuniculus and Mus musculus are shown. Secondary structure elements are displayed above the sequences. The residues of the IZUMO1–JUNO interface are indicated by yellow highlighting. The sequence identity between human and mouse IZUMO1 and JUNO are 59% and 69%, respectively, for the extracellular regions used in this study. In contrast, the interface residues exhibit less conservation (35% and 53% identity for IZUMO1 and JUNO, respectively). The N-glycosylation sites are indicated by blue Y-shaped characters. Alignments were performed using Clustal Omega software (EMBL-European Bioinformatics Institute). Residues are coloured to indicate the degree of similarity: red residues are those with the highest similarity, followed by green, blue, and black (lowest similarity).

Extended Data Figure 2 Sequence alignment of JUNO and folate receptor (FR).

Sequence alignment of JUNO from Homo sapiens, Equus caballus and Mus musculus and FRα, FRβ, and FRγ from Homo sapiens. Secondary structure elements for JUNO and FR are displayed above and below the sequences, respectively. The residues involved in the folate binding in FRs are indicated by yellow highlighting. Alignments were performed using Clustal Omega software (EMBL – European Bioinformatics Institute). Residues are coloured to indicate the degree of similarity: red residues are those with the highest similarity, followed by green, blue, and black (lowest similarity).

Extended Data Figure 3 Anomalous difference Fourier maps from S-SAD data.

The anomalous difference Fourier maps from the data collected at 2.7 Å wavelength contoured with green at the 3σ level are superposed onto the refined model of the IZUMO1–JUNO complex. The disulfide bonds are shown in stick representations and indicated by green (IZUMO1) and cyan (JUNO) labels.

Extended Data Figure 4 Superpositions of IZUMO1 and JUNO structures.

a, Superposition of the six IZUMO1 molecules (chains A–F) in the asymmetric unit. b, Superposition of the two (form 1; chains A and B) and four (form 2; chains A–D) JUNO molecules in the asymmetric unit. c, Superposition of IZUMO1 (chain A) and JUNO (chain A) structures onto the IZUMO1–JUNO complex structure (form 1, form 2, and form 3). IZUMO1 (chain A) and JUNO (chain A) are coloured grey. The IZUMO1–JUNO complexes in the form 1, form 2, and form 3 (chain A–B and chain C–D) crystals are coloured green, cyan, orange, and magenta, respectively. The r.m.s.d. values for each pair of molecules are shown schematically.

Extended Data Figure 5 Cell surface expression of IZUMO1 mutants in COS-7 cells (related to Fig. 4).

Surface localization of wild-type IZUMO1 or its mutant in COS-7 cells. IZUMO1 proteins on the cell surface (green) and nuclei (blue) were stained. Scale bars, 20 μm.

Extended Data Figure 6 Dimerization of IZUMO1 and IZUMO1–JUNO complex analysed by SV–AUC and size-exclusion chromatography.

a, b, The oligomerization states of IZUMO1 (a) and IZUMO1–JUNO complex (b) were analysed by SV–AUC at various concentrations. The normalized c(s) distributions were plotted against the sedimentation coefficients, s20,w (S). The observed sedimentation coefficient of the IZUMO1 dimer (3.2 S) at a high concentration (100 μM) in a was smaller than the expected value for IZUMO1 dimer (3.7 S), owing to the fast monomer–dimer interconversion kinetics. c, The oligomerization of IZUMO1 was analysed by gel-filtration chromatography. In each experiment, 20, 100 or 740 μM IZUMO1 (total volume of 50 μl) was injected into a Superdex 200 Increase 5/150 GL gel-filtration column (running buffer; 10 mM Tris-HCl pH 7.5 and 150 mM NaCl).

Extended Data Figure 7 Effect of reducing agent and acidic pH on the IZUMO1–JUNO interaction.

a, DTT-induced aggregation of IZUMO1. Each sample was incubated at room temperature for 2 h in the presence of 1 mM DTT, and then injected into a Superdex 200 Increase 5/150 GL gel-filtration column (running buffer; 10 mM Tris-HCl pH 7.5 and 150 mM NaCl, 1 mM DTT). Eluents were analysed by SDS–PAGE. For gel source data, see Supplementary Fig. 1. b, The interaction between IZUMO1 and JUNO at acidic pH. The IZUMO1–JUNO interaction at acidic pH was analysed by size-exclusion chromatography (SEC; top) and isothermal titration calorimetry (bottom) as in Fig. 1. In SEC analysis, each sample was injected into a Superdex 200 Increase 5/150 GL gel-filtration column (running buffer; 10 mM MES-NaOH pH 5.5 and 150 mM NaCl). Eluents were analysed by SDS–PAGE. c, The SEC chromatograms at a neutral pH of 7.5 is shown as a control experiment.

Extended Data Table 1 Isothermal titration calorimetry results
Full size table
Extended Data Table 2 Crystallization and cryoprotectant conditions
Full size table
Extended Data Table 3 Data collection and refinement statistics
Full size table

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This file contains the uncropped blots with size marker indications. (PDF 655 kb)

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Ohto, U., Ishida, H., Krayukhina, E. et al. Structure of IZUMO1–JUNO reveals sperm–oocyte recognition during mammalian fertilization. Nature 534, 566–569 (2016). https://doi.org/10.1038/nature18596

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