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

The first step in mammalian fertilization, where both species specificity and the one-sperm-per-egg norm are enforced, is recognition between coat proteins in the membrane around the egg (the zona pellucida) and sperm. The mouse zona pellucida protein ZP3 is the primary receptor for sperm and its most conserved domain (ZP-N) is also found in many different extracellular proteins with a wide range of biological functions. The crystal structure of the ZP-N domain of ZP3 has now been determined. ZP-N adopts an immunoglobulin-like fold but shares little similarity with other immunoglobulin-like domains, marking it out as a new subtype of this superfamily. As well as providing a glimpse of mammalian fertilization at atomic resolution, the structure of ZP3 ZP-N is of relevance for reproductive medicine and also for diseases such as non-syndromic deafness or renal and vascular disorders, where ZP-N containing proteins are involved. Species-specific recognition between the egg extracellular matrix (zona pellucida) and sperm is the first step of mammalian fertilization. This paper reports the 2.3 A resolution structure of the 'zona pellucida filament' of the egg, which act as sperm receptors. The structure supports the presence of ZP–N repeats within the amino-terminal region of ZP2 and other vertebrate zona pellucida/vitelline proteins, and has implications for egg coat architecture and the post-fertilization block to polyspermy and speciation. 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 angstrom (A) 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.