In moths, pheromone-binding proteins (PBPs) are responsible for the transport of the hydrophobic pheromones to the membrane-bound receptors across the aqueous sensillar lymph. We report here that recombinant Antheraea polyphemus PBP1 (ApolPBP1) picks up hydrophobic molecule(s) endogenous to the Escherichia coli expression host that keeps the protein in the "open" (bound) conformation at high pH but switches to the "closed" (free) conformation at low pH. This finding has bearing on the solution structures of undelipidated lepidopteran moth PBPs determined thus far. Picking up a hydrophobic molecule from the host expression system could be a common feature for lipid-binding proteins. Thus, delipidation is critical for bacterially expressed lipid-binding proteins. We have shown for the first time that the delipidated ApolPBP1 exists primarily in the closed form at all pH levels. Thus, current views on the pH- Chemoreception in insects is mediated via sensing of a variety of small, volatile organic compounds. Chemoreception plays a critical role not only in the regulation of the most fundamental chemosensory behaviors in insects but also for intraspecies communication. Pheromone is a chemical signal that triggers a natural behavioral response in another member within the same species. In lepidopteran insects, sex pheromones produced by females are detected by males of the same species with extreme sensitivity and selectivity. These hydrophobic compounds are transported to the membrane-bound receptors (ion channels) (1, 2) across the aqueous sensillar lymph by the pheromone-binding proteins (PBPs).2 PBPs are small, acidic proteins, highly soluble in water, with a molecular mass of 14 -16 kDa. The first PBP to be identified, cloned, and expressed in the bacterial system was that from the giant silk moth Antheraea polyphemus (3, 4). Since then, PBPs have been isolated from at least eight moth species that share about 50% sequence identity, with six conserved cysteine residues forming three disulfide bridges that are important for the formation of the hydrophobic binding pocket (5).Moth PBPs are known to undergo a dramatic conformational switch with a change in pH, which has been proposed to be necessary for the release of ligand at lower pH near the membrane-bound receptors (ion channels) (6 -11). The current view on insect PBPs is that the unliganded protein exists in two conformations as follows: form A (PBP A ), the acidic or "ligand releasing" conformation that exists at low pH, and form B (PBP B ), the basic or "ligand binding" conformation that exists at high pH (7). The low and high pH conformations have been determined by solution NMR for PBPs from two representative moths, Bombyx mori (BmorPBP) and Antheraea polyphemus (ApolPBP1) (8 -11), which are believed to represent the "free" or "unliganded" form of the two proteins. High pH conformations of both "ligand-bound" and free forms of BmorPBP have also been determined by x-ray crystallography (12, 13).The conformation of BmorPBP B in solution at pH 6.5 (9) cons...

Pheromone-binding proteins (PBPs) in lepidopteran moths selectively transport the hydrophobic pheromone molecules across the sensillar lymph to trigger the neuronal response. Moth PBPs are known to bind ligand at physiological pH and release it at acidic pH while undergoing a conformational change. Two molecular switches are considered to play a role in this mechanism: (i) Protonation of His70 and His95 situated at one end of binding pocket, and (ii) Switch of the unstructured C-terminus at the other end of the binding pocket to a helix that enters the pocket. We have reported previously the role of the histidine-driven switch in ligand release for Antheraea polyphemus PBP1 (ApolPBP1). Here we show that the C-terminus plays a role in ligand release and binding mechanism of ApolPBP1. The C-terminus truncated mutants of ApolPBP1 (ApolPBP1ΔP129-V142 and ApolPBP1H70A/H95AΔP129-V142) exist only in the bound conformation at all pH levels, and they fail to undergo pH- or ligand- dependent conformational switch. Although these proteins could bind ligands even at acidic pH unlike the wild-type ApolPBP1, they had ~4 fold reduced affinity towards the ligand at both acidic and physiological pH than that of ApolPBP1wt and ApolPBP1H70A/H95A. Thus, apart from helping in the ligand-release at acidic pH, the C-terminus in ApolPBP1 also plays an important role in ligand binding and/or locking the ligand in the binding pocket. Our results are in stark contrast to those reported for BmorPBP and AtraPBP, where C-terminus truncated proteins had similar or increased pheromone-binding affinity at any pH.

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The lectin isolated from mature seeds of Cicer arietinum (CAL) agglutinates pronase-treated rabbit and human erythrocytes and its haemagglutination activity is inhibited by fetuin and desialated fetuin but not by simple monosaccharides or oligosaccharides. The puri®ed lectin is a dimer of molecular weight 43 000 Da composed of two identical subunits (MW 21 500), as con®rmed by SDS±PAGE. The lectin has been crystallized using the hangingdrop vapour-diffusion method at 295 K over a well solution containing 0.2 M sodium acetate, 0.1 M sodium phosphate buffer pH 6.5 and 14%(w/v) polyethylene glycol 8000. The triangular prism-shaped crystals belong to space group R3 and have unit-cell parameters a = b = 81.2, c = 69.4 A Ê . The diffraction data are 93.8% complete to 2.3 A Ê Bragg spacing with an R merge of 0.103.

Based on their carbohydrate specificity, the jacalin family of lectins can be divided into two groups: galactose-specific and mannose-specific. The former are cytoplasmic proteins, whereas the latter are localized in the storage vacuoles of cells. It has been proposed that the post-translational modification in some of the lectins that splits their polypeptide chains into two may be crucial for galactose specificity. The mannose-specific members of the family are single-chain proteins that lack the above modification. Although the galactose-specific and the mannose-specific jacalin-type lectins differ in their sequences, they share a common fold: the beta-prism I fold, which is characteristic of Moraceae plant lectins. Here, two crystal structures of a jacalin-related lectin from Artocarpus hirsuta, which is specific for galactose, in complex with methyl-alpha-D-galactose are reported. The lectin crystallized in two orthorhombic forms and one hexagonal form under similar conditions. The crystals had an unusually high solvent content. The structure was solved using the molecular-replacement method using the jacalin structure as a search model. The two orthorhombic forms were refined using data to 2.5 and 3.0 A resolution, respectively. The structures of the A. hirsuta lectin and jacalin are identical. In orthorhombic form I the crystal packing provides three different micro-environments for sugar binding in the same crystal. The observed difference in the specificity for oligosaccharides between the A. hirsuta lectin and jacalin could only be explained based on differences in the molecular associations in the packing and variation of the C-terminal length of the beta-chain. The observed insecticidal activity of A. hirsuta lectin may arise from its similar fold to domain II of the unrelated delta-endotoxin from Bacillus thuringiensis.