9513421 Stout The goal of this project is to apply methods of molecular biology and x-ray crystallography in concert to understand molecular events controlling and occurring during fertilization. A dual approach is being taken: to study in depth structure function relationships in sperm lysin, for which two high resolution crystal structures, an expression system and a quantitative biological assay are all available; and to initiate and carry out structure determinations of three additional sperm and egg proteins. To understand key steps during fertilization it is necessary to solve the crystal structures of the molecules involved. The proteins under investigation are involved in critical steps of fertilization. The only crystal structures of a fertilization protein available are of the monomer and dimer of sperm lysin. Recently, an expression system has been developed which yields milligrams of very pure and biologically active material. An outstanding opportunity now exists to understand the function of this fertilization protein. These crystallographic experiments will enable cell biologists to visualize and understand fertilization at the molecular level. Four proteins are under investigation: 16K and 18K sperm lysins, ADP-ribosyl cyclase and sperm protein 56 (sp56). sp56 mediates specific sperm-egg interaction in the mouse. Abalone sperm 16K lysin dissolves a hole in the egg vitelline envelope (VE) permitting the sperm to penetrate the VE. Fusion of the sperm and egg cytoplasmic membranes is promoted by 18K lysin. Cyclase from Aplysia ovotestis synthesizes cyclic ADP-ribose (cADPR) from NAD. cADPR regulates the release of intracellular Ca2+ and induction of the cortical reaction, resulting in the formation of the fertilization envelope. A model has been developed for the mechanism of VE dissolution by lysin. It proposes specific functions for the novel surface features of the protein both in the monomeric state, as it binds to the VE, and in t he dimeric state, as it exists in solution and within the acrosomal granule. All aspects of the hypothesis for the mechanism of VE dissolution can be tested with site-directed mutagenesis. Mutant proteins will be assayed for biological activity and their crystal structures solved. These experiments promise to define for the first time the activity of a fertilization protein at the level of individual amino acids. Diffraction quality crystals have been obtained of ADP-ribosyl cyclase. Crystallographic analysis of ADP-ribosyl cyclase will provide the first insight into cADPR-receptor interaction and provide a basis for modeling the catalytic mechanism of the enzyme. Crystals have been obtained of green abalone 18K lysin, and recombinant sp56 will be used for crystallization experiments. ***
