Kinetics and molecular properties of pheromone binding and release

Abstract

Transient kinetic studies have shown that the uptake of the pheromone (bombykol) of the silkworm moth (Bombyx mori), by its pheromone-binding protein (PBP) BmorPBP, proceeds with an ''on'' rate of 0.068 ؎ 0.01 M ؊1 ⅐s ؊1 . With the high concentration of PBP in the sensillar lymph (10 mM), the half-life for the uptake of pheromone in vivo is Ϸ1 ms. A pH-dependent conformational change (BmorPBP B 3 BmorPBP A ), associated with the release of pheromone, is a first-order reaction (k ‫؍‬ 74.1 ؎ 0.32 s ؊1 ; t1/2, 9.3 ms). Under physiological conditions, both reactions proceed with half-life times on the order of milliseconds, as is required for odorant-oriented navigation in insects. Molecular interactions of bombykol with both native and mutated PBPs were analyzed by a novel binding assay. A recombinant protein with the native conformation (BmorPBP) showed high binding affinity (K D ‫؍‬ 105 nM) at pH 7 but low affinity (K D ‫؍‬ 1,600 nM) at pH 5, when tested at both low and high KCl concentrations. A protein with a C-terminal segment deleted (BmorPBP⌬P129-V142) was found to bind bombykol at pH 7 and at pH 5 with the same affinity as the native protein at pH 7, indicating that the C-terminal segment is essential for preventing binding at low pH. Binding studies with three mutated proteins (BmorPBPW37F, BmorPBPW127F, and BmorPBPW37A) showed that replacing Trp-37 (with Phe or Ala) or Trp-127 (with Phe) did not affect the binding affinity to bombykol. Fluorescence studies shed light on the contributions of Trp-37 and Trp-127 emissions to the overall fluorescence.Bombyx mori pheromone-binding protein ͉ bombykol ͉ effect of C terminus on pheromone release ͉ fast uptake of pheromone and delivery ͉ mutated pheromone-binding proteins F or many insects, small-molecule signals communicate the availability of food, the presence of friends and foes, and the readiness to mate. In general, mate-finding is an essential prerequisite for exploring insects' enormous reproductive potential and, consequently, leads to their domination of the terrestrial world. To advertise their readiness to mate, female moths, for example, produce and release sex pheromones. Only minute amounts are released, so as to avoid chemical conspicuousness. Once released, the chemical signals are diluted in the environment and mixed with a myriad of physiologically irrelevant compounds, including pheromones from other species. Even though each species communicates with a specific pheromonic language, males can detect the low-level signals from conspecific females because of a highly developed olfactory system. To find females and successfully reproduce, males may have to make long-distance, odorant-oriented flights. Navigation requires a highly selective, sensitive, and dynamic sensory system for the detection of pheromones. Given the structure of pheromone plumes (1), insects have only a few milliseconds to reset their detectors while flying in the clean air spaces between pockets of chemical signals.Pheromones are largely hydrophobic compounds, whereas pheromo...

Crystal and solution structures of an odorant-binding protein from the southern house mosquito complexed with an oviposition pheromone

“…At a pH lower than 5.5, the C terminus of BmorPBP adopts a newly formed α-helix conformation occupying the bombykol-binding site, whereas at a pH higher than 5.5, the same C-terminus region forms an elongated stretch outside the binding site, making it available for the pheromone. These results led the authors to propose a pH-induced ligand-releasing mechanism, through which BmorPBP ejects the bound pheromone to its receptor when encountering the low pH generated by the negatively charged membrane surfaces (15,(18)(19)(20)(21)(22). A similar ligand-releasing mechanism is observed for a PBP from the giant silk moth Antheraea polyphemus (14,23).…”

“…Octanal, nonanal, decanal, and geranylacetone showed significant binding affinity, but γ-octalactone did not bind. and quantified by gas chromatography according to a previously reported "cold-binding assay" (20). Additionally, binding was measured by a competitive binding assay using N-phenyl-1-naphthylamine as a fluorescent reporter (47).…”

Niemann–Pick type C2 protein mediating chemical communication in the worker ant

“…OBP is able to bind its ligand at neutral pH and to eject it in a very fast process (t 1/2 = 9 ms) in low pH condition localized on adjacent negatively charged olfactory receptor neuron (28,29). Our binding assay showed that CjapNPC2 bound to a wide variety of long-chain fatty acids, alcohols, and acetates at neutral pH and dissociated with these ligands at low pH.…”

Comparative genomics of chemosensory protein genes reveals rapid evolution and positive selection in ant-specific duplicates

“…Growing evidence supports that odorant binding proteins (OBP) and chemosensory proteins (CSP) are involved in solubilizing and transporting odorants and pheromones through aqueous hemolymph in insects (Leal et al, 2005;Ozaki et al, 2005). The chemical messages carried by the OBPs and CSPs are decoded when odorant receptors (OR) or in some cases gustatory receptors (GR) selectively bind different chemicals (Vosshall and Stocker, 2007).…”