Sequence - Structure – Function: Analysis of the Takeout Protein Superfamily Using D. Melanogaster Courtship as a Model System

Courtship behavior is a well characterized and extensively studied complex innate behavior in D. melanogaster adult males. Our lab has previously established the role of the secreted protein Takeout (To) in courtship behavior. To is a small soluble 23kD protein encoded by a gene that belongs to a family of 23 paralogs in D. melanogaster. To is sex-preferentially expressed in the fat body surrounding the brain in male flies. Homologs of To have been documented in other species as well. The relationship between these family members, however, remains unclear. Here, in this work I show that paralogs CG13618 and CG16820 can rescue the courtship behavior of to1 mutants. I also show that a To ortholog from A. aegypti (Diptera) and a homolog from the Light Brown Moth Epiphyas postvittana (Lepidoptera) can also rescue the courtship defects in to1 mutants. In this study I have also established the functional significance of conserved residues in motif-2 of the To protein sequence. Using site-directed mutagenesis I show that on mutating the conserved residues in motif-2 to Alanines, To only partially retains its function. Further, I also show that the hemolymph Juvenile Hormone Binding Protein (hJHBP) from Silkworm (Bombyx mori), which has very low sequence identity (31%identity, 34% sequence coverage) can partially substitute for To. I have used protein structure modeling programs to examine the sequences and structural features of the rescuing proteins and identified residues that might be critical in the ligand binding by To. Taken together my experiments establish that distant To homologs from other species and within D. melanogaster can functionally substitute for To in courtship behavior, despite their limited sequence similarities. Moreover, hJHBP from Bombyx mori can partially substitute for To suggesting that although To and hJHBPs have diverged and belong to two separate protein families they have partially retained their function. Partial rescue of courtship behavior by hJHBP also suggests that hJHBPs might be able to bind the same ligand as To.