Conformational Change in the Pheromone-binding Protein fromBombyx mori Induced by pH and by Interaction with Membranes

Abstract

The pheromone-binding protein (PBP) from Bombyx mori was expressed in Escherichia coli periplasm. It specifically bound radiolabeled bombykol, the natural pheromone for this species. It appeared as a single band both in native and SDS-polyacrylamide gel electrophoresis and was also homogeneous in most chromatographic systems. However, in ion-exchange chromatography, multiple forms sometimes appeared. Attempts to separate them revealed that they could be converted into one another. Analysis of the protein by circular dichroism and fluorescence spectroscopy demonstrated that its tertiary structure was sensitive to pH changes and that a dramatic conformational transition occurred between pH 6.0 and 5.0. This high sensitivity to pH contrasted markedly with its thermal stability and resistance to denaturation by urea. There was also no significant change in CD spectra in the presence of the pheromone. The native protein isolated from male antennae displayed the same changes in its spectroscopic properties as the recombinant material, demonstrating that this phenomenon is not an artifact arising from the expression system. This conformational transition was reproduced by interaction of the protein with anionic (but not neutral) phospholipid vesicles. Unfolding of the PBP structure triggered by membranes suggests a plausible mechanism for ligand release upon interaction of the PBP-pheromone complex with the surface of olfactory neurons. This pH-linked structural flexibility also explains the heterogeneity reported previously for B. mori PBP and other members of this class of proteins.Recognition of chemical signals in insects takes place in their olfactory sensilla. A class of small proteins (ϳ13-17 kDa), odorant-binding proteins (OBPs), 1 is believed to facilitate the passage of hydrophobic odorant molecules from the environment to the surface of olfactory receptor neurons (1-3). These proteins are present in the sensillar lymph at an enormous 10 -20 mM concentration (3). In Lepidoptera, two classes of OBPs have been distinguished: one involved in the recognition of sex pheromones (pheromone-binding proteins (PBPs)) and another thought to participate in the recognition of general odorants (general odorant-binding proteins (GOBPs)) (4). Since the identification of the first such proteins in Antheraea polyphemus (5), homologous proteins have been detected and characterized in many species from several insect orders. A number of OBPs have been cloned (6 -14), and a few of them have been expressed (15-18). Sequences of lepidopteran PBPs are highly conserved, but their homology to GOBPs and OBPs from other insect orders is only moderate. The whole family, however, shows highly conserved motifs, among them the six cysteine residues thought to participate in formation of disulfide bonds (6). Circular dichroism measurements and theoretical structure prediction have revealed that these proteins are in a large part ␣-helical (15, 19). However, despite substantial efforts in the past several years, their three-dimensional struct...

New Insights into the Mechanism of Odorant Detection by the Malaria-transmitting Mosquito Anopheles gambiae

“…The 1 H, 15 N HSQC spectrum of apoOBP1 recorded at pH 7.4 contains 100 of an expected 116 peaks, indicating that the protein is relatively well ordered (supplemental Fig. S3A).…”

“…S1A). To probe tertiary structure formation, we recorded two-dimensional 1 H, 15 N HSQC NMR spectra. In the spectrum of apoOBP4 we observe only 35 intense, well resolved peaks from backbone amides of an expected 114 (Fig.…”

“…We obtained backbone chemical shift assignments for all but three of the major peaks in the 1 H, 15 N HSQC spectrum of OBP1 recorded in the presence of indole. Residues that could not be assigned are located predominantly in helices 5 and 6 (blue, Fig.…”

“…There is evidence that OBPs have two primary roles in odorant perception. In the first model a number of groups have proposed that OBPs act as passive carriers for the odorant, and pH changes in the vicinity of the dendritic membrane lead to conformational changes that stimulate ligand release, freeing the ligand to activate the receptor (13)(14)(15)(16)(17). In support of this, Carlson and coworkers (18,19) have shown that in Drosophila, many odorant receptors, when expressed in "empty" neurons, exhibit the same response profile to odorants as the wild-type receptors, providing evidence that the odorant itself directly activates the receptor.…”

Kinetics and molecular properties of pheromone binding and release

“…Upon interaction with negatively charged sites on or near the receptors, the PBP-pheromone complex undergoes a conformational change that leads to the ejection of the pheromone (3)(4)(5). In the PBP from the silkworm moth, Bombyx mori (BmorPBP), this pH-dependent conformational change (4,5,8) leads to the formation of a C-terminal ␣-helix in the acidic form of the protein (BmorPBP A ) (5). The newly formed ␣-helix in Bmor-PBP A occupies the cavity that serves as a binding pocket in the basic form of the protein (BmorPBP B ).…”