The pheromone binding protein of B. mori has six helices, and bombykol binds in a completely enclosed hydrophobic cavity formed by four antiparallel helices. Bombykol is bound in this cavity through numerous hydrophobic interactions, and sequence alignments suggest critical residues for specific pheromone binding.

Odorants are transmitted by small hydrophobic molecules that cross the aqueous sensillar lymph surrounding the dendrites of the olfactory neurons to stimulate the olfactory receptors. In insects, the transport of pheromones, which are a special class of odorants, is mediated by pheromone-binding proteins (PBPs), which occur at high concentrations in the sensillar lymph. The PBP from the silk moth Bombyx mori (BmPBP) undergoes a pH-dependent conformational transition between the forms BmPBP A present at pH 4.5 and BmPBP B present at pH 6.5. Here, we describe the NMR structure of BmPBP A , which consists of a tightly packed arrangement of seven ␣-helices linked by well defined peptide segments and knitted together by three disulfide bridges. A scaffold of four ␣-helices that forms the ligand binding site in the crystal structure of a BmPBP-pheromone complex is preserved in BmPBP A . The C-terminal dodecapeptide segment, which is in an extended conformation and located on the protein surface in the pheromone complex, forms a regular helix, ␣7, which is located in the pheromone-binding site in the core of the unliganded BmPBP A . Because investigations by others indicate that the pH value near the membrane surface is reduced with respect to the bulk sensillar lymph, the pH-dependent conformational transition of BmPBP suggests a novel physiological mechanism of intramolecular regulation of protein function, with the formation of ␣7 triggering the release of the pheromone from BmPBP to the membrane-standing receptor. Male moths have an exquisitely sensitive olfactory system capable of detecting over great distances single molecules of pheromones emitted by the female, and they are capable of distinguishing highly selectively between closely similar compounds or isomers (1). These capabilities of male moths are because of olfactory sensory organs that consist of large branched antennae and can readily be isolated for biochemical characterization or electrophysiological recordings (2). The surface of the antennae is covered with hairlike protrusions composed of cuticle, which form a sheath (sensillum) surrounding one or several dendrite endings from olfactory neurons. The dendrites in each sensillum are bathed in a special fluid, the sensillum lymph. Odorant molecules gain access to the dendritic membrane through pores penetrating the sensillum wall and then crossing the sensillar lymph to interact with G proteincoupled olfactory receptors, which form a large eukaryotic protein family. Upon interaction with the olfactory receptors, a cascade of events leads to a change of the membrane potential (3), resulting in opening of ion channels and depolarization.Common odorants are hydrophobic molecules that are poorly soluble in aqueous media, such as the sensillar lymph. Odorants overcome this barrier by binding to odorant-binding proteins (OBPs). OBPs are present at high concentrations in the lymph (up to 10 mM), bind the odorant, and transport it from the sensillum pore wall to the receptor at the dendritic membrane. It is ...

Disulfide bond formation is the only known posttranslational modification of insect pheromone binding proteins (PBPs). In the PBPs from moths (Lepidoptera), six cysteine residues are highly conserved at positions 19, 50, 54, 97, 108 and 117, but to date nothing is known about their respective linkage or redox status. We used a multiple approach of enzymatic digestion, chemical cleavage, partial reduction with Tris-(2-carboxyethyl)phosphine, followed by digestion with endoproteinase Lys-C to determine the disulfide connectivity in the PBP from Bombyx mori (BmPBP). Identification of the reaction products by on-line liquid chromatography-electrospray ionization mass spectrometry (LC/ESI-MS) and protein sequencing supported the assignment of disulfide bridges at Cys-19-Cys-54, Cys-50-Cys-108 and Cys-97-Cys-117. The disulfide linkages were identical in the protein obtained by periplasmic expression in Escherichia coli and in the native BmPBP.z 1999 Federation of European Biochemical Societies.

The nuclear magnetic resonance structure of the unliganded pheromone-binding protein (PBP) from Bombyx mori at pH above 6.5, BmPBP B , consists of seven helices with residues 3^8, 16^22, 29^32, 46^59, 70^79, 84^100, and 107^124, and contains the three disul¢de bridges 19^54, 50^108, and 97117. This polypeptide fold encloses a large hydrophobic cavity, with a su⁄cient volume to accommodate the natural ligand bombykol. The polypeptide folds in free BmPBP B and in crystals of a BmPBP^bombykol complex are nearly identical, indicating that the B-form of BmPBP in solution represents the active conformation for ligand binding.