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.
Highlights
Sperm–egg fusion is the critical step in mammalian fertilization, and requires the interaction between IZUMO1 on the sperm surface and JUNO ( known as folate receptor (FR) 4 or IZUMO1R) on the egg surface
Mouse JUNO has two putative N-glycosylated sites (Asn[73] and Asn[185]; Fig. 1a), and the glycosylation may hinder the crystallization. This notion is consistent with the fact that the WT ectodomain of mouse JUNO, which was expressed in human embryonic kidney 293S (HEK293S) GnT1 À cells and enzymatically deglycosylated, was successfully crystallized[13]
A previous functional analysis suggested that flexible regions 1 and 3 around the central pocket are involved in IZUMO1 binding[13]
Summary
Sperm–egg fusion is the critical step in mammalian fertilization, and requires the interaction between IZUMO1 on the sperm surface and JUNO ( known as folate receptor (FR) 4 or IZUMO1R) on the egg surface. JUNO binds IZUMO1 and is involved in the sperm–egg adhesion[3], whereas the FRs bind folate and participate in folate uptake[7,8,9,10]. The crystal structures of FR1 and FR2 revealed that the FRs adopt a globular architecture stabilized by multiple disulfide bonds and recognize folate in a folate-binding pocket[11,12]. The structure revealed conformational differences in three loop regions between JUNO and the FRs, despite they have similar overall structures. To gain mechanistic insights into the IZUMO1 recognition by JUNO, we solved the crystal structure of the N73D mutant of mouse JUNO and performed the structure-based mutational analyses
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