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

Abstract

Species-specific recognition between the egg extracellular matrix (zona pellucida) and sperm is the first, crucial step of mammalian fertilization. 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 infertile. 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' module, whose conserved amino-terminal part (ZP-N) was suggested to constitute a domain of its own. 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 (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 beta-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.

A structured interdomain linker directs self-polymerization of human uromodulin

“…An additional C 5′ -C 6′ disulfide tethers EGF IV C317 to ZP-N C347, which belongs to an α-helix that lies between strands B and C (Fig. 2B) and is absent in ZP3 (3,5). Loss of either Cys is also associated with ADTKD, due to intracellular aggregation and impaired urinary secretion of UMOD (22,23) (Fig.…”

“…Mass spectroscopy (MS) analysis of ZP-C disulfide linkages suggests that there are two types of ZP modules with different structures (13). Type II contains 10 conserved Cys (C 1-7,a,b,8 ) and both homopolymerizes (UMOD, GP2, and TECTA) and heteropolymerizes (ZP1, ZP2, and ZP4), whereas type I (ZP3) includes eight conserved Cys (C [1][2][3][4][5][6][7][8] ) and only heteropolymerizes with type II (7,13,14). However, MS studies of egg coat protein disulfides are contradictory (15), and type II disulfide linkages C 5 -C 6 , C 7 -C a , and C b -C 8 are compatible neither with the fold of ZP3 (3) nor with structures of the ZP-C domain of BG, whose ZP module contains 10 Cys (16,17).…”

Proteomics enhances evolutionary and functional analysis of reproductive proteins

“…The ZP domain of each protein contains two distinct portions: the N terminus, ZP-N, is responsible for polymerization of ZP proteins into fibrils, while the C terminus, ZP-C, has regions that regulate secretion and, in the case of ZP2 and ZP3, sperm binding. The crystal structure of the ZP-N domain of ZP3 was recently solved, (99) providing detail into the formation of ZP structures and raising the possibility of understanding, at the molecular level, why particular mutations in ZP proteins lead to infertility. Curiously, ZP2 has multiple ZP-N domains preceding a single ZP-C domain, and these domains may function in preventing polyspermy and maintaining the species specificity of fertilization.…”

“…Curiously, ZP2 has multiple ZP-N domains preceding a single ZP-C domain, and these domains may function in preventing polyspermy and maintaining the species specificity of fertilization. Using the ZP3 ZP-N structure to comparatively model the ZP2 ZP-N domains (99) and examining the structural effects of changes in positively selected sites should enhance our understanding of sperm-egg recognition and coevolution.…”

Zona pellucida glycoprotein ZP3 and fertilization in mammals

“…Each ZP glycoprotein has a characteristic region ($260 amino acids) known as the ZP domain that also is present in proteins related to ZP1-3 that make up the extracellular coat (vitelline envelope) of fish, frog, and bird eggs (Jovine et al, 2005;Litscher and Wassarman, 2007). The ZP domain is a bipartite structure and the N-terminal half of the polypeptide serves as a polymerization module (Jovine et al, 2004Monn e et al, 2008). ZP and vitelline envelope proteins have been conserved for more than 600 million years.…”