Structural studies of proteins essential for fertilization

Where do babies come from? Even though it sounds like a fairly obvious question, it took more than 200 years to discover that sperm, whose first observation dates back to the 17th century, can penetrate into the oocyte and fuse with it. Interestingly, this initial study was carried out in starfish, and since then, scientists have addressed the fascinating question of how gamete recognition functions using very different model systems. Egg-sperm binding is a species-specific event, and it is thought to require binding proteins that are exposed on the surface on the egg and sperm. In this thesis, we focus our attention on how egg coat subunits are involved in this process and investigate it using X-ray crystallography and biochemistry. The mammalian egg coat, also known as Zona Pellucida (ZP), is a thick extracellular matrix that surrounds the egg and is made of glycoproteins (known as ZP1-4) that assemble into long cross-linked filaments. Although ZP3 has been identified as a sperm receptor, it remains unclear as to how it interacts with sperm at molecular level. Paper I explores the potential role of sperm protein 56 (sp56) as a receptor for the egg coat protein ZP3. Its putative role as a ZP3-binding partner has been previously assessed by several in vitro assays using soluble proteins. Here, we express the recombinant form of hamster sp56 and attempt to reconstitute a hetero-complex with recombinant ZP3. Sp56 is secreted as a homo-hexamer held together by intramolecular disulfides. Considering the high avidity conferred by the homo-oligomeric state of the glycoprotein, the inability to observe any interaction to ZP3 by either pull-down or Far western blotting (Far WB) indicates that very-low affinity binding events might mediate egg-sperm interaction. Different from the mammalian scenario, egg and sperm proteins in marine invertebrates are known to bind more tightly. Paper II sheds light on the event occurring when gametes first meet using abalone as a model system. We discover that egg coat functional units in mammals and invertebrates are structurally conserved and, remarkably, despite millions of years of evolution they rely on a common ZP-N fold domain to accomplish their functions. Crystallographic studies on individual subunits of the egg coat receptor VERL (vitelline envelope receptor for lysin) in complex with the sperm protein lysin, provides the first snapshot of egg-coat recognition and clarifies how species-specificity is maintained. By combining structural and biochemical data, the paper proposes the mechanistic model of how sperm penetrates the egg coat at the molecular level. For structural studies of egg coat glycoproteins, it is essential to produce correctly folded material that is stable, highly homogeneous and is secreted in large amounts. To achieve this, we fuse glycoproteins to maltose binding protein (MBP) and describe in Paper III the advantages of such a method using mammalian and insect cell expression systems. The study herein shows how fusion to MBP boosts the expression of six challenging glycoproteins that are poorly secreted if unfused. MBP not only improves crystallization but provide initial phasing information for structural determination by molecular replacement. Given the relevant role of egg coat subunits in fertilization, we investigate in Paper IV their functional relationship to two specular aspects of reproductive health. The study provides detailed information on how an anti-ZP-reagent recognizes its epitopes, and investigates how mutations in ZP genes relate to female infertility. By doing so, our findings further strengthen the importance of ZP in the development of novel contraceptive strategies and provide a better understanding of the impact of ZP mutations on human fertility