A Structural and Functional Investigation of Germ Plasm Organization Mediated by D. Melanogaster Oskar Protein

Abstract

Germ cells are the unique source of gametes for multicellular organisms and are specified through different mechanisms. One such specification mechanism is inheritance-mediated, in which the molecular factors necessary and sufficient to impart germ cell fate (collectively referred to as “germ plasm”) are maternally synthesized and deposited during oogenesis or early embryogenesis. Incorporation of this germ plasm into newly formed cells results in primordial germ cells. This mechanism stands in contrast to the predominant metazoan (and likely ancestral) mode of germ cell specification termed induction; germ cells specified via induction are the result of extracellular signaling from one embryonic tissue to another, triggering the expression of germ plasm components in the recipient cell type. In Drosophila melanogaster, the gene oskar (osk) is necessary and sufficient for organizing germ plasm in the oocyte, leading to primordial germ cell specification. While many investigators have offered thorough developmental and genetic investigations of osk, an understanding of the specific molecular mechanisms by which its protein products accumulate germ plasm is presently lacking. Oskar protein is uncharacterized, but is predicted to contain two well-folded domains: an N-terminal winged helix-turn-helix domain (the LOTUS/OST-HTH domain) and a C-terminal domain bearing sequence similarity to SGNH hydrolases (the lipase-related domain). The function of both domains is unknown, but previously published mutational data demonstrate that the lipase-related domain is crucial for Osk protein function. It is currently not known if the LOTUS/OST-HTH domain is necessary for the accumulation of germ plasm at the posterior pole. Using X-ray crystallography, I solved four structures of the LOTUS domain (corresponding to residues 139-241) and found that it forms a homodimer. In contrast to published characterized winged helix-turn-helix structures, the LOTUS homodimer conformation results from two β-hairpins (one from each protomer) forming a completed β-sheet. Analytical size exclusion chromatography, bacterial two-hybrid, and multi-angle light scattering experiments confirm that the LOTUS domain exists as a dimer in solution and combined with site-directed mutagenesis, the dimerization interface is indeed formed by a completed β-sheet as revealed by the crystal structures. Using a GAL4/UAS inducible D. melanogaster transgenic osk reporter, deletion of the LOTUS domain was found to abrogate the accumulation of germ plasm components, indicating that this domain is necessary for Oskar protein function. Furthermore, substituting the LOTUS domain with exogenous dimerization domains does not restore Osk activity. It is then postulated that oligomerization as mediated by the LOTUS domain constitutes at least one crucial aspect of its function within the context of full length Oskar protein.