Final Report Summary - GAMETE RECOGNITION (Molecular Basis of Mammalian Egg-Sperm Interaction) In all sexually reproducing organisms, the interaction between male and female gametes – the sperm and the egg - marks the beginning of a new life. This crucial biological event involves two distinct adhesion steps, one taking place when sperm contacts the extracellular coat of the egg, and the other when sperm fuses with the plasma membrane of the oocyte after having penetrated the egg coat. Both steps are mediated by the formation of intermolecular complexes between counterpart gamete proteins, but detailed information on the corresponding interfaces has been elusive. The goal of this project was to determine the molecular basis of fertilization by using structural biology to visualize the architecture of the egg coat, establish how this binds sperm, and understand how this interaction is prevented after gamete fusion. Towards these objectives, we not only investigated molecules directly involved in fertilization, but also studied a selection of biomedically important human proteins that have different biological functions, but share structural similarity with egg coat components. Moreover, in order to obtain milligram amounts of functionally active molecules for both biochemical and structural studies, we developed a mammalian expression system based on fusion to a mammalianized version of bacterial maltose-binding protein.From marine invertebrates to human, a conserved family of glycoproteins generates the egg coat by forming micron-long extracellular filaments that are cross-linked into a three-dimensional matrix. Polymerization of egg coat subunits depends on a common C-terminal zona pellucida (ZP) module, which consists of two structurally related immunoglobulin (Ig)-like domains (ZP-N and ZP-C). By determining the structure of several ZP modules with different polymerization ability, we suggested that, although all these modules share the same overall fold, their ability to form filaments – and whether these filaments are homo- or heteropolymeric – depends on the linker sequence between ZP-N and ZP-C. Of direct relevance for this process, which is activated upon subunit processing at a consensus cleavage site, was also our collaborative involvement in the identification of the first protease that specifically cuts a ZP module substrate. Parallel structural studies of the N-terminal regions of the egg coat proteins, which have been implicated both in filament cross-linking and sperm binding, revealed that they also adopt a ZP-N fold. This suggests that the basic architecture conserved in all egg coats is the result of multiple duplication events of an ancestral gene that encoded an Ig-like molecule. Importantly, this finding also unifies invertebrate and vertebrate fertilization by unveiling a previously unappreciated structural similarity between the sperm-interacting regions of egg coat proteins from mollusk to human.Building on these results, we recently succeeded in obtaining atomic-level information on how two cognate egg-sperm recognition proteins contact each other at the beginning of fertilization. This work has revealed an an intimate structural connection between egg coat recognition and penetration by sperm at fertilization. Finally, by determining the structure of egg plasma membrane-associated protein Juno, we have provided the first detailed information on a molecule essential for triggering gamete fusion in mammals.Taken together, the studies supported by this project started to shed light on the molecular basis of fertilization, an essential biological process that has been fascinated mankind for centuries but whose detailed mechanism remained unknown. Information deriving from this work will hopefully contribute to the future of reproductive medicine by suggesting new ways to address infertility and contraception.
