Structural and functional insights into IZUMO1 recognition by JUNO in mammalian fertilization

Abstract

Sperm-egg fusion is the critical step in mammalian fertilization, and requires the interaction between IZUMO1 on the sperm surface and JUNO (also known as folate receptor (FR) 4 or IZUMO1R) on the egg surface. Whereas other FRs bind and uptake folates, JUNO binds IZUMO1 and establishes the cell-cell adhesion. However, the mechanism of IZUMO1 recognition by JUNO has remained elusive. Here we report the crystal structure of mouse JUNO, at 2.3 Å resolution. A structural comparison of JUNO with the FRs revealed that JUNO and the FRs have similar overall structures, but JUNO lacks the folate-binding pocket, thereby explaining the inability of JUNO to bind folate. Further complementation of Juno knockout eggs with mutant Juno messenger RNAs revealed that the conserved, surface-exposed tryptophan residue of JUNO is required for sperm binding and fertilization. Our structure-based in vivo functional analyses provide a framework towards a mechanistic understanding of mammalian gamete recognition.

Figure 1. Crystal structure of JUNO.

(a) Multiple sequence alignment of JUNOs from different species. In mouse JUNO, the two glycosylation sites (Asn73 and Asn185) and the eight conserved disulfide bonds are indicated by blue and yellow triangles, respectively. Trp62 is indicated by a gold triangle. Residues forming the central pocket are indicated by red triangles. (b) Ribbon representation of the N73D mutant ectodomain of mouse JUNO (stereo view). Flexible regions 1–3 are coloured green, blue and orange, respectively. The disulfide bonds and N-linked glycans are shown as stick models, and the disordered regions are shown as dashed lines. Trp62 is shown as a gold stick. (c) Asn185-linked glycan. The Asn185-linked glycan and the side chains of Asn146 and Leu148 are depicted as stick and space-filling models. (d) Ribbon representations of the ectodomain of WT JUNO (PDB code 5EJN). Mol A (left) and Mol B (right) are shown as in b. (e) Superimposition of our N73D mutant JUNO (coloured) and WT JUNO (PDB code 5EJN) (grey).

Figure 2. Comparison of JUNO with the FRs.

(a) Multiple sequence alignment of mouse JUNO, human FR1 and human FR2. The FR1 residues involved in folate binding are indicated by grey triangles. The other key residues are indicated as in Fig. 1a. (b,c) Ribbon representation of the human FR1–folate complex (PDB code 4LRH) (b) and N73D mutant JUNO (c). (d,e) Molecular surface (d) and folate-binding pocket (e) of the human FR1–folate complex (PDB code 4LRH). (f,g) Molecular surface (f) and central pocket (g) of N73D mutant JUNO. (h) Superimposition of the central pocket of N73D mutant JUNO and the folate-binding pocket of FR1 (stereo view). Trp184 of JUNO is highlighted as a semi-transparent space-filling model.

Figure 3. IZUMO1-binding site.

(a) Schematic showing the sperm–egg fusion complementation assay. After mRNA injection and in vitro maturation, Hoechst 33342 was loaded to visualize the MII chromosome and the fused sperm heads. (b) Functional complementation of Juno KO eggs by mRNA injection. The mRNA encoding the WT or mutants of mouse JUNO (mJUNO), or human JUNO (hJUNO), was injected into the mouse Juno KO eggs. The fusion of mouse spermatozoa was visualized by the transfer of DNA dye from the egg to the sperm nuclei. The extrusion of the second polar body was used as an indicator for successful fertilization. Asterisks indicate unfertilized eggs. Scale bar, 50 μm. (c) Conservation of the surface residues of the N73D mutant JUNO. The sequence conservation among JUNOs from 14 mammalian species (human, monkey, gorilla, chimpanzee, elephant, pig, horse, bear, cat, rabbit, golden hamster, mouse, rat and opossum) was calculated using the ConSurf server (http://consurf.tau.ac.il), and is coloured from cyan (low) to maroon (high).

Figure 4. Sperm binding to JUNO-expressing cultured cells.

(a) Z-stack confocal microscopy image of seeded cells and inseminated Red-IZUMO1 spermatozoa. Red-IZUMO1, mJUNO and DNA (Hoechst 33342) signals are indicated in red, green and blue, respectively, in the merged image. The heads of spermatozoa bound to mJUNO-expressing HEK293T cells are indicated with arrowheads. Scale bar, 20 μm. (b) Binding of spermatozoa with mitochondrial DsRed2 to HEK293T cells expressing WT mJUNO, mutant mJUNOs or hJUNO. Light (upper image) and fluorescence (lower image) microscopic images of transfected cells after insemination with fluorescent spermatozoa are shown. Each picture corresponds to a quarter of the area of the field used for analysis. Scale bar, 200 μm. (c) Sperm-binding abilities of JUNO-expressing HEK293T cells. The number of spermatozoa bound to HEK293T cells expressing WT mJUNO was set to 1.0. Data are the mean±s.d. Asterisks indicate significant differences (Student's t-test; *P<0.05 and ^**P<0.005).

Figure 5. Putative IZUMO1-binding surface.

(a,b) A putative IZUMO1-binding site encompassing Trp62 and flexible regions 1 and 3 is indicated by a circle on the ribbon (a) and surface (b) representations of N73D mutant JUNO.