Structure of betaglycan zona pellucida (ZP)-C domain provides insights into ZP-mediated protein polymerization and TGF-β binding

Abstract

The zona pellucida (ZP) domain is a bipartite protein structural element comprised of ZP-N and ZP-C regions. Most notable for its ability to mediate protein polymerization, many ZP proteins polymerize and assemble into long fibrils that form specialized extracellular matrices. Other ZP proteins (namely, betaglycan and endoglin) do not polymerize but serve as important membrane coreceptors for ligands in the transforming growth factor-β (TGF-β) superfamily. Here, we present the 2.0-Å resolution crystal structure of the betaglycan ZP-C region in combination with a downstream region known as the external hydrophobic patch (EHP). Similar to the ZP-N region, the ZP-C region also adopts an immunoglobulin-like fold, despite sharing no sequence homology and possessing different disulfide linkages. The EHP region, which was previously thought to be external to the ZP region, is integral to the ZP-C domain and corresponds to the ZP-C G strand. Our structure also indicates that the critical maturation cleavage of ZP proteins, a process that activates nascent ZP proteins for polymerization, occurs within the immunoglobulin domain at the FG loop. Nonpolymerizing ZP proteins such as betaglycan and endoglin do not contain this cleavage site. Finally, our structure suggests that the AB loop and the convex surface pocket are regions important for TGF-β ligand binding.reproductive biology | signaling | inhibin T he zona pellucida (ZP) domain is a structural element found in the extracellular region of many eukaryotic proteins (1, 2). This structural element is characterized by a set of eight highly conserved cysteines and predicted to exist as a bipartite structure corresponding to ZP-N and ZP-C regions tethered by a linker (3, 4). The crystal structure of the ZP-N region has been solved and shown to adopt an immunoglobulin-like fold (5), suggesting that the ZP-C region is likely also a distinct domain by itself and that the so-called ZP domain, the collective structural element comprised of distinct ZP-N and ZP-C domains, may be more aptly referred to as the ZP region.The ZP region is important for protein polymerization and protein-protein interactions (6, 7). Many ZP proteins polymerize to become long fibrils that form specialized extracellular matrices. The best-known example is the pericellular matrix known as zona pellucida or vitelline envelope that surrounds mammalian and nonmammalian oocytes, respectively, to ensure speciesspecific fertilization and prevent polyspermy. This matrix is composed from homologues of ZP1, ZP2, ZP3, and in some species, ZP4 proteins, all of which contain a single ZP region in the ectodomain (1). In Drosophila melanogaster, two ZP proteins, Piopio and Dumpy, contribute to the apical extracellular matrix and play crucial roles in tracheal development and tubulogenesis (8). In mammals, other well-characterized polymerizing ZP proteins include uromodulin (also known as Tamm-Horsfall protein) that likely forms the water-impermeable layer around the thick ascending loop of Henle in the kidney (9) and...

A structured interdomain linker directs self-polymerization of human uromodulin

“…S3 and S8). Remarkably, these combinations coincide with the different polymerization abilities of the corresponding proteins: BG and ENG do not polymerize; ZP1-ZP4 and TECTB heteropolymerize; and UMOD, GP2, and TECTA homopolymerize (7,17,37,38). This observation is consistent with the idea that in the last set of proteins, coupling of an α1/β1-containing linker to ZP-C induces an extended conformation of the ZP module.…”

“…Surprisingly, our crystallographic data reveal that UMOD ZP-C (Figs. S1D and S5A) also shares the same fold and disulfide connectivity of ZP3 and BG ZP-Cs (3,16,17) (Fig. S5 B and C), except for the C a -C b disulfide not found in ZP3 proteins (15) (Fig.…”

“…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 1-8 ) 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). At the same time, interpretation of the latter data in relation to polymerization is complicated by the fact that, like ENG, BG remains membrane-associated and does not form filaments (7,17).…”

“…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). At the same time, interpretation of the latter data in relation to polymerization is complicated by the fact that, like ENG, BG remains membrane-associated and does not form filaments (7,17).To gain insights into the mechanism of ZP module protein assembly, we carried out X-ray crystallographic studies of the complete polymerization region of UMOD. The structure reveals that a rigid interdomain linker is responsible for maintaining UMOD in a polymerization-competent conformation.…”

Involvement of Carbohydrate Residues of the Zona Pellucida in In Vitro Sperm Recognition in Pigs and Cattle

“…Porcine ZP3 and ZP4 exhibit disulfi de patterns different from the ZP3 and ZP1/ZP2/ZP4 patterns reported for mice, rats, humans, and fi sh (Kanai et al 2008 ). The chick homologue of the mammalian ZP3 precursor protein has a pig-type ZP3 pattern (Han et al 2010 ), whereas betaglycan has a pig-type ZP1/ZP2/ZP4 pattern (Lin et al 2011 ), as revealed by X-ray crystallography. The two ZP3 disulfi de bond pattern s cause only subtle structural differences (Han et al 2010 ).…”