Crystal Structure of Epiphyas postvittana Takeout 1 with Bound Ubiquinone Supports a Role as Ligand Carriers for Takeout Proteins in Insects

Cyril Hamiaux,Duncan Stanley,Dave R Greenwood,Edward N Baker,Richard D Newcomb

doi:10.1074/jbc.m807467200

Abstract

Takeout (To) proteins are found exclusively in insects and have been proposed to have important roles in various aspects of their physiology and behavior. Limited sequence similarity with juvenile hormone-binding proteins (JHBPs), which specifically bind and transport juvenile hormones in Lepidoptera, suggested a role for To proteins in binding hydrophobic ligands. We present the first crystal structure of a To protein, EpTo1 from the light brown apple moth Epiphyas postvittana, solved in-house by the single-wavelength anomalous diffraction technique using sulfur anomalous dispersion, and refined to 1.3 angstroms resolution. EpTo1 adopts the unusual alpha/beta-wrap fold, seen only for JHBP and several mammalian lipid carrier proteins, a scaffold tailored for the binding and/or transport of hydrophobic ligands. EpTo1 has a 45 angstroms long, purely hydrophobic, internal tunnel that extends for the full length of the protein and accommodates a bound ligand. The latter was shown by mass spectrometry to be ubiquinone-8 and is probably derived from Escherichia coli. The structure provides the first direct experimental evidence that To proteins are ligand carriers; gives insights into the nature of endogenous ligand(s) of EpTo1; shows, by comparison with JHBP, a basis for different ligand specificities; and suggests a mechanism for the binding/release of ligands.

Highlights

Behavior and as such may represent novel targets for insect control strategies
There are, two major differences between the families: a second disulfide bond in the juvenile hormone-binding proteins (JHBPs), not present in To; and the presence of two conserved C-terminal sequence motifs that are unique to the To family (2)
In the light brown apple moth Epiphyas postvittana (Tortricidae: Lepidoptera), a common horticultural pest, we identified an abundant To protein (EpTo1) that is more highly expressed in male when compared with female antennae (17)

Summary

Introduction

Behavior and as such may represent novel targets for insect control strategies. The original member, To, was discovered in Drosophila melanogaster as a clock-regulated gene, acting as a molecular link between circadian rhythms and feeding behavior (1, 2). Ligand-binding Tunnel and Bound Surrogate Ligand—The most striking feature of the EpTo1 structure is the large central tunnel, ϳ45 Å long; beginning with a slightly wider cavity at the top of the molecule, it extends for the entire length of the barrel (Fig. 2C).

Results

Conclusion