The c-terminus of the protein bears mostly non-polar 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 <ref>doi: 10.1016/j.bbrc.2005.07.176</ref>. Of these, residues <scene name='68/683383/Bombykol_ligand_in_2p71/2'>Asp-132</scene> (and Glu-141, if present) triggers the formation of the alpha-helix upon protonation at low pH. This causes the <scene name='68/683383/1dqe_2fjy-bom/1'>ejaculation of the ligand from the binding pocket</scene>, which is replaced by the formatted alpha helix<ref>doi: 10.1016/j.bbrc</ref>.
The c-terminus of the protein bears mostly non-polar 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 <ref>doi: 10.1016/j.bbrc.2005.07.176</ref>. Of these, residues <scene name='68/683383/Asp132/1'>Asp-132</scene> (and Glu-141, if present) triggers the formation of the alpha-helix upon protonation at low pH. This causes the <scene name='68/683383/1dqe_2fjy-bom/1'>ejaculation of the ligand from the binding pocket</scene>, which is replaced by the formatted alpha helix<ref>doi: 10.1016/j.bbrc</ref>.
Odorant-binding protein (OBP) are soluble proteins which involve in the processes of odorant detection in the olfactory sensilla [1]
Though functionally same, vertebrates and insects OBP have different origin and structure.
OBPs are important for insect olfaction. For instance, OBP76a (LUSH) in the fly Drosophila melanogaster is required for the detection of the pheromone vaccenyl acetate [2] and has been proven to adopt a conformation that activates the odorant receptor [3].
Bombyx mori, the silk moth, picture by Fernando CuencaBombykol, a sex pheromone of Bombyx mori, from PubChem
OBP in insects
OBP Function
Despite five decades of intensive research, the exact roles of OBP and the mechanism by which the odorant receptor (OR) is activated are still in dispute [4][5].
A few functions have been suggested for OBP:
1. Solubelizing the odorant molecule and its transportation in the sensillar lymph.
2. Protecting the odorant molecule from the odorant degrading enzymes, in the sensillar lymph.
3. Activating of the odorant receptor on the dendrite membrane, by the odorant-OBP complex.
4. Mediating the deactivation of the odorant molecule after the activation of the receptor.
5. An organic anion (the protein has 9 negative charges).
Of all, the first role of OBP as an odorant solubilizer and carrier is generally accepted.
In order to explain the structure and function of these fascinating proteins, this page will further focus on a particular OBP - the well investigated Bombyx mori PBP: BmorPBP.
Pheromone binding proteins (PBPs) are specialized members of the insect odorant-binding protein (OBP) super-family.
The main purpose 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 journey begins when the female moth releases a sex pheromone, usualy in specific hours in the night [1].
BmorPBP was first identified in the B. mori male antennae by Krieger et al. in 1996 [2], as the PBP of the first sex pheromone discovered ((E,Z)-10,12-hexadecadienol, or Bombykol). The male moth needs to detect minute 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[3]).
The protein has 164 amino acids that forms 6-7 alpha helices (depends on the protein conformation). Three disulfide bonds formed by 6 cystein residues tied four helices, and form the compact and robust structure of the protein. As expected from a soluble protein, its surface is covered with charged residues, which allows it to make interactions with the water molecule and solubilize in the sensillar lymph.
BmorPBP - ligand binding
The protein natural ligand is the moth pheromone Bombykol. However, it was demonstrated that other molecules can also bound to the protein cavity [4]. The interaction with the ligand is being made by 4 alpha helices 1, 4, 5 and 6 in the core of the protein, which form the binding cavity [5].
Inside the binding cavity, non-charged residues 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 hydrogen bond with the sidechain of Ser56, 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 "closed form" (A) and the "open form" (B)[6]. 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), whereas in the "open form" (A) the c-terminus is outside of the protein and has no defined secondary structure. Binding experiments have shown that the B-form binds 15 times higher than the A-form [7], 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.
The transition between the two conformation is both pH and ligand dependent [8][9][10]. In short, the B-form (c-terminus outside the cavity) occurs only at neutral pH and in the presence of the ligand. The A-form (c-terminus inside the cavity) occurs at both low and neutral pH, yet at the latter only in the absence of ligand. Therefore, in neutral pH when the ligand is binding to the protein in its A-form, the complex formation causes a change in conformation to the B-form. However, both A and B forms are equally distributed in the lymph.
Conformation transition mechanism:
The c-terminus of the protein bears mostly non-polar 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, if present) triggers the formation of the alpha-helix upon protonation at low pH. This causes the ejaculation of the ligand from the binding pocket, which is replaced by the formatted alpha helix[12].
Studies on other Lepidopterans that show a similar pH dependent conformation suggests that this model is a general model moth PBP[13].
Nonetheless, the enormous diversity among insects is not allowing us to assume this model is true for all insects' OBPs.
Figure 1. The events prior the neuron excitation, following the "N model" suggested by Kaissling (2009)[14]: The pheromone enters the sensillar lymph through a pore in cuticle. The pheromone can then be degraded by the ODE (1) -or- bind to the A and B protein forms (2a and 2b, respectively). When the complex arrives at the low pH near the membrane, the transition is in favor of the A-form, (3) in which the -c-terminus is forming an alpha helix inside the binding cavity, pushing out the pheromone. The activation of the complex of odorant receptor and coreceptor (OR:OR-CO), is induced by ether the complex of pheromone-PBP, or by the pheromone alone (5, two options). The B-form can also act as a scavenger, as it mediates the deactivation of the pheromone (6) and releases it to the ODE (6)
Receptor activation
Two theories have been proposed 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.
Activation by the pheromone alone
This model is supported by the pH dependent conformation transition, that is described above. The bulk of the sensillar lymph is in neutral 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 [15], which cause the accumulation of positively charged kations near the membrane surface (20-50 nm)[14]. 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 Drosophila, where it was found that the complex of pheromone-PBP is required for the activity of pheromone-sensitive neurons <ref>DOI: 10.1016/j.neuron.2004.12.031Cite error: The opening <ref> tag is malformed or has a bad name<ref>doi: 10.1016/j.cell.2008.04.046Cite error: The opening <ref> tag is malformed or has a bad name
↑J. Krieger, E. von Nickisch-Rosenegk, M. Mameli, P. Pelosi, H. Breer. Binding proteins from the antennae of Bombyx mori. Insect Biochemistry and Molecular Biology, Volume 26, Issue 3, March 1996, Pages 297-307 doi:https://dx.doi.org/10.1016/0965-1748(95)00096-8
↑Vogt, Richard G., Riddiford, Lynn M. Pheromone binding and inactivation by moth antennae. Nature 293, 161 - 163 (10 Sep 1981) doi:https://dx.doi.org/10.1038/293161a0
↑Lautenschlager C, Leal WS, Clardy J. Bombyx mori pheromone-binding protein binding nonpheromone ligands: implications for pheromone recognition. Structure. 2007 Sep;15(9):1148-54. PMID:17850754 doi:10.1016/j.str.2007.07.013
↑Benjamin H Sandler, Larisa Nikonova, Walter S Leal, Jon Clardy. Sexual attraction in the silkworm moth: structure of the pheromone-binding-protein�bombykol complex, Chemistry & Biology (2000), Vol 7, Issue 2, 143-151 doi:https://dx.doi.org/10.1016/S1074-5521(00)00078-8
↑Hubert Wojtasek and Walter S. Leal. Conformational Change in the Pheromone-binding Protein fromBombyx mori Induced by pH and by Interaction with Membranes, 1999 The Journal of Biological Chemistry, 274, 30950-30956. doi:https://dx.doi.org/10.1074/jbc.274.43.30950
↑Leal WS, Chen AM, Ishida Y, Chiang VP, Erickson ML, Morgan TI, Tsuruda JM. Kinetics and molecular properties of pheromone binding and release. Proc Natl Acad Sci U S A. 2005 Apr 12;102(15):5386-91. Epub 2005 Mar 22. PMID:15784736 doi:https://dx.doi.org/10.1073/pnas.0501447102
↑Lautenschlager C, Leal WS, Clardy J. Coil-to-helix transition and ligand release of Bombyx mori pheromone-binding protein. Biochem Biophys Res Commun. 2005 Oct 7;335(4):1044-50. PMID:16111659 doi:10.1016/j.bbrc.2005.07.176
↑Damberger FF, Michel E, Ishida Y, Leal WS, Wuthrich K. Pheromone discrimination by a pH-tuned polymorphism of the Bombyx mori pheromone-binding protein. Proc Natl Acad Sci U S A. 2013 Nov 12;110(46):18680-5. doi:, 10.1073/pnas.1317706110. Epub 2013 Oct 24. PMID:24158483 doi:https://dx.doi.org/10.1073/pnas.1317706110
↑Lautenschlager C, Leal WS, Clardy J. Coil-to-helix transition and ligand release of Bombyx mori pheromone-binding protein. Biochem Biophys Res Commun. 2005 Oct 7;335(4):1044-50. PMID:16111659 doi:10.1016/j.bbrc.2005.07.176
↑Cite error: Invalid <ref> tag; no text was provided for refs named Leal
↑ 14.014.1Cite error: Invalid <ref> tag; no text was provided for refs named Kaissling 2009
↑Thomas A Keil, Surface coats of pore tubules and olfactory sensory dendrites of a silkmoth revealed by cationic markers, Tissue and Cell (1984), Vol 16, Issue 5, 705-717 doi:https://dx.doi.org/10.1016/0040-8166(84)90004-1
↑Pelosi P, Iovinella I, Felicioli A, Dani FR. Soluble proteins of chemical communication: an overview across arthropods. Front Physiol. 2014 Aug 27;5:320. doi: 10.3389/fphys.2014.00320. eCollection, 2014. PMID:25221516 doi:https://dx.doi.org/10.3389/fphys.2014.00320
↑Xu P, Atkinson R, Jones DN, Smith DP. Drosophila OBP LUSH is required for activity of pheromone-sensitive neurons. Neuron. 2005 Jan 20;45(2):193-200. PMID:15664171 doi:10.1016/j.neuron.2004.12.031
↑Laughlin JD, Ha TS, Jones DN, Smith DP. Activation of pheromone-sensitive neurons is mediated by conformational activation of pheromone-binding protein. Cell. 2008 Jun 27;133(7):1255-65. PMID:18585358 doi:10.1016/j.cell.2008.04.046
↑Leal WS. Odorant reception in insects: roles of receptors, binding proteins, and degrading enzymes. Annu Rev Entomol. 2013;58:373-91. doi: 10.1146/annurev-ento-120811-153635. Epub, 2012 Sep 27. PMID:23020622 doi:https://dx.doi.org/10.1146/annurev-ento-120811-153635
↑Kaissling KE. Olfactory perireceptor and receptor events in moths: a kinetic model revised. J Comp Physiol A Neuroethol Sens Neural Behav Physiol. 2009 Oct;195(10):895-922. , doi: 10.1007/s00359-009-0461-4. Epub 2009 Aug 21. PMID:19697043 doi:https://dx.doi.org/10.1007/s00359-009-0461-4
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