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

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 Å resolution. EpTo1 adopts the unusual ␣/␤-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 Å 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. Takeout (To) 3 family have been proposed to have important roles in various aspects of insect physiology and 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). In addition, To expression is regulated by the somatic sex determination pathway and affects male courtship behavior (3, 4). To has a predicted secretion signal (1) and is secreted in the hemolymph of adult males (4). At least 80 To genes have now been identified from seven orders of insect, including 20 from D. melanogaster, but few have been characterized in any detail. Due to the localization and/or expression profiles of their transcripts, some are proposed to have roles in chemosensory perception (5-8), whereas others are thought to be involved in binding terpenoids (9), including juvenile hormones (JH) (10 -15). Members of theTo proteins share limited sequence similarity and a conserved N-terminal disulfide bond with juvenile hormone-binding proteins (JHBPs). These proteins specifically bind and transport JH to target tissues in the Lepidoptera, further suggesting a role for To proteins in binding hydrophobic ligands (2). There are, however, two major differences between the families: a second disulfide bond in the JHBPs, not present in To; and the presence of two conserved C-terminal sequence motifs that are unique to the To family (2). Very recently, the structure of JHBP from the moth Galleria mellonella (GmJHBP) has been solved, revealing an unusual fold in which a highly curved antiparallel ␤-sheet wraps around a...

Conserved SMP domains of the ERMES complex bind phospholipids and mediate tether assembly

“…SMP-containing proteins are localized predominantly at contact sites between the ER and other organelles (18). They belong to the tubular lipid-binding protein (TULIP) superfamily comprising known lipid-binding proteins such as Takeout (19), the bactericidal/permeability-increasing protein (BPI) (20), the cholesteryl ester transfer protein (CETP) (21), and the lipopolysaccharide-binding protein (LBP) (22). The crystal structure of the SMP domain of the human extended synaptotagmin 2 protein (E-SYT2) was determined recently (23), revealing its tubular structure with a hydrophobic tunnel occupied by a phospholipid (Fig.…”

Precise RNAi-mediated silencing of metabolically active proteins in the defence secretions of juvenile leaf beetles

“…It possesses a conserved domain (pfam06585) characteristic for the juvenile hormone-binding protein (JHBP) superfamily. Sequence comparisons using the BLAST algorithm [35] revealed that the P. cochleariae amino acid sequence shares only very limited identity with functionally characterized insect proteins, for example, 12 per cent identity with the JHBP from Bombyx mori [43] and 16 per cent with the takeout (To) 1 from Epiphyas postvittana [44] (figure 2a). Higher identities up to 25 per cent were found only with insect proteins not yet fully characterized in their functions, such as those with the To-like protein (NP_001191952) from Acyrtosyphon pisum or the JHBP-like (XP_001359416) from Drosophila pseudoobscura pseudoobscura.…”

“…Several lines of evidence indicate that JHBPs form complexes with juvenile hormones (JHs) which provide protection of the chemically labile JHs against nonspecific enzymatic degradation and/or adsorption to lipophilic surfaces during the delivery process from the production site to the target tissue [46,47,49]. Up to now only the crystal structure of To 1 from E. postvittana with ubiquinone provided direct evidence for ligand binding in To proteins [44]. Most of the putative To proteins await elucidation of their mode of action.…”

“…In his work, the stereospecifity of the cyclization was analysed and allocated to an enzymatic conversion. However, to date JHBPs and To proteins have been established as being carriers of hydrophobic ligands [44,48]. Several lines of evidence indicate that JHBPs form complexes with juvenile hormones (JHs) which provide protection of the chemically labile JHs against nonspecific enzymatic degradation and/or adsorption to lipophilic surfaces during the delivery process from the production site to the target tissue [46,47,49].…”

Automated identification of crystallographic ligands using sparse-density representations

“…soaking) crystallization experiments, identification of the ligands giving rise to difference electron density following macromolecular model building is generally facile. However, it is less straightforward when small molecules, typically endogenous substrates or effectors that adhere to the protein during expression, remain bound during purification and crystallization (Hamiaux et al, 2009;Girardi et al, 2010) or when multiple ligands are added to crystals simultaneously. The latter approach may improve efficiency in fragment-based drug design (Mooij et al, 2006) and in metabolite cocktail screening for identification of protein function (Shumilin et al, 2012).…”