Odorant binding protein

Pheromone binding proteins (PBPs) are specialized members of the insect odorant-binding protein (OBP) super-family.

The main purpse in the dult moth's short life is reproduction. In fact, the male and female moth invest all of theire energy and resourses hoping to reach to the ultimate goal- mating. This long journy begins when the female moth releases a sex pheromone, usualy in specific hours in the night ^[3]. BmorPBP was first identified in the B. mori male antennae by Krieger et al. in 1996 ^[4], as the PBP of the first sex pheromone discovered ((E,Z)-10,12-hexadecadienol, or [Bombykol]). The male moth needs to detect minut amount of the pheromone in the air, while following turbulent wind-born pheromone trail and response fast (experimental evidence shows a response time of 0.5 seconds^[5]). As the electrical signal transduction after the activation of the odorant receptor are too fast, Kaissling ^[6]have suggested that the

BmorPBP structure and function

The protein has 164 amino acids that forms 6-7 alpha helices. Three disulfide bonds (in yellow) formed by residues tied four helices, and form the compact and robust sturcture of the protein. As expected from a soluble protein, its surface is covered with , which allows it to make interactions with the water molecule and sollubilize in the sensillar lymph.

BmorPBP ligand and ligand binding

The protein natural ligand is the moth pheromone . However, it was demonstrated that other molecules can also bound to the protein cavity ^[7]. The interaction with the ligand is beeing made by 4 alpha helices 1, 4, 5 and 6 in the core of the protein, which form the binding cavity ^[8]. Inside the binding cavity, are interacting with the pheromone, mainly by van der waals bounds. Out of those residues, some are conserved across OBP of lepidopteran (in green), and the rest are conserved in lepidopteran PBP only (in light blue). In addition, the hydroxyl group of the pheromone bombykol forms a , Ser56 in red, oxygens are in purple (O–O distance of 2.8 Å).

Protein conformations

The A and B forms without ligand (PDB IDs: 1gm0 and 1ls8).

BmorPBP has two conformations: The "open form" (A) and the "close form" (B)^[9]. The bombykol and the alpha-helix loacated in the c-terminus of the protein compete for the binding site: when the c-terminus is inside the binding cavity it get's an alpha helix shape, and the protien is in its "close form" (B), wherease in the "open form" (A) the c-terminus is outside of the protein and has no defined secondery sturcture. Binding experiments have shown that the B-form binds 15 times higher than the A-form ^[10], therefore considered to be the carrier of the pheromone. The complex of the A-form and the pheromone, is then considered the form that activates the receptor.

In short, the B-form (c-terminus outside the cavity) occurse only at neutral pH and in the presence of the ligand. The A-form (c-terminus inside the cavity) occurs at both low and nuetral pH, yet at the latter only in the absence of ligand. Therfor, in neutarl pH when the ligand is binding to the protein in its A-form, the complex formation causes a change in conforamtion to the B-form.

The transition between the two conformation is taking place between 6-5 pH.

Conformation transition mechanism:

The c-terminus of the protein bears mostly nonpolar amino acids. Yet on the surface of the helix there are three exceptional amino acids: Asp-132, Glu-137, and Glu-141, which are conserved in moth PBP ^[11]. Of these, residues Asp-132 and Glu-141 triggers the formation of the α-helix upon protonation at low pH. This what is causing the ejacullate of the ligand from the binding pocket, which is replaced by the formated alpha helix^[12].

The is both pH and ligand dependent ^[13][14][15].

Studies on other Lepidopterans that show a simmilar pH dependent conforamation suggests that this model is a genral model moth PBP^[1]. Nonetheless, the enourmas diversity among insects is not allowing us to assume this model is true for all insects' OBPs.

Receptor activation

Two theories have been propsed for the activation of the odorant receptors located on the dendrtirte membrane. One theory suggests that the pheromone-PBP complex is needed for the receptor activation, while the second theory argue that the pheromone itself is sufficient for the activation of the receptor.

The events prior the neuron excitation, following the "N model" suggested by Kaissling (2009)^[6]

• Activation by the pheromone alone

This model is supported by the pH dependent conforamtion transition, that is described above. The bulk of the sensillar lymph is in nuetral pH (6.5-7), while environment near the dendrite membrane bears a low pH (4.5), due to the negative charges on the surface of the membrane ^[16], which cause the accumulation of positively charged kations near the membrane surface (20-50 nm)^[6]. According to this model (illustrated in figure 1), the pheromone is entering the sensillar lymph through a pore in the cuticle, then it can be either degraded by odorant degrading enzymes (ODE) or bind to a PBP (of both forms). Once the complex is arriving to the low pH environment near the dendrite membrane the PBP will shift to the A-form, thereby ejaculating the ligand from the binding pocket, allowing it to activate the OR:CO-OR complex and the cellular signal transduction begins.

• Activation by the complex pheromone-PBP

An alternative mode of action was proposed for the receptor activation in the drosophila, where it was found that the complex of pheromone-PBP is required for the activity of pheromone-sensitive neurons ^[17]