Sex at the molecular level: The crystal structures of abalone fertilization proteins

Abalone are broadcast spawners that exhibit species specific fertilization even though many of the species have overlapping breeding seasons and habitats. The head of the abalone sperm possesses a large acrosomal granule containing roughly equal amounts of two proteins, a 16 kDa protein named lysin, and an 18 kDa protein named sp18. Contact between the sperm and the egg vitelline envelope (VE) triggers the acrosome reaction, which involves the exocytotic release of both proteins and the elongation (by actin polymerization) of the acrosomal process. The dimeric lysin is deposited onto the surface of the egg VE where it binds to the VE receptor for lysin (VERL) and creates a hole in the VE by a species-specific, non-enzymatic mechanism. Sp18 coats the membrane covering the acrosomal process where it is thought to mediate sperm and egg cell membrane fusion. Lysin and sp18 arose by gene duplication from an ancestral protein that was capable of both VE dissolution and plasma membrane fusion. Both proteins have diverged substantially, and although lysin retains the ability to mediate membrane fusion, sp18 is unable to dissolve the VE.; As part of an effort to understand, at atomic resolution, the interactions between the sperm and the egg, I solved the x-ray crystal structures of lysin and sp18 from Haliotis fulgens (green abalone) as well as the high-resolution structure of lysin from Haliotis rufescens (red abalone). The lysin molecules from both species of abalone consist of homodimers with large clefts that may act as binding sites for VERL. Each lysin subunit contains a 5-helix bundle with two basic tracks running the length of one side and a solvent exposed hydrophobic patch on the other side. The overall folds of the two lysins are remarkably similar, suggesting that species-specificity resides in the locations of non-conserved residues and in subtle side-chain rearrangements. Sp18 has the same 5-helix motif, but occurs as a monomer rather than a dimer and lacks the hydrophobic patch and basic tracks found in lysin. Instead, sp18 contains large areas of basically-charged and hydrophobic surface as well as a ridge of acidic residues. The increased hydrophobic surface found on sp18 accounts for its fusagenic activity, while the absence of the dimeric structure and the difference in surface charge may explain sp18's inability to bind to VERL and dissolve the VE. I have also made significant progress in the purification and expression of red abalone VERL in order to biochemically characterize its interaction with lysin and eventually solve its crystal structure.