The authors have declared that no competing interests exist.
Conceived and designed the experiments: EdL WL DW. Performed the experiments: EdL YI DW. Analyzed the data: EdL WL DW. Contributed reagents/materials/analysis tools: EdL YI WL DW. Wrote the paper: EdL WL DW.
Current address: Department of Applied Biosciences, Kyungpook National University, Daegu, South Korea
Current address: Biotechnology Research Center and Department of Biotechnology, Toyama Prefectural University, Imizu, Toyama, Japan
The navel orangeworm,
Larvae from the navel orangeworm,
Understanding the structural basis of pheromone detection and elucidating the mechanisms of action of olfactory proteins will aid in the development of novel and stable mimics of one or more of the attractants. These pathways are mediated in part by pheromone-binding proteins (PBPs), which are believe to be important in solubilizing the hydrophobic ligands and serve as an initial transporter that present odorants to a receptor in the membrane to elicit an olfactory response. Some PBPs bind pheromones with high affinity at the relatively high ambient pH present in the sensillar lymph and release them in the lower pH and cationic conditions, which are thought to exist near the membrane
A primary pheromone-binding protein (PBP) from
A pET22-b(+) vector containing DNA encoding mature AtraPBP1 was used to transform BL21 (DE3) competent cells (EMD Chemicals, Novagen, Gibbstown, NJ). The transformant was used to inoculate LB medium containing carbenicilin and cells were cultured at 200 rpm at 28°C overnight. After IPTG induction for 3 hours, the recombinant protein was extracted from harvested cells using a freeze-thaw procedure. The recombinant AtraPBP1 was purified by a combination of ion-exchange chromatography and gel-filtration as described previously
Purified AtraPBP1 was crystallized at room temperature by the hanging drop vapor diffusion method. Drops composed of 1 µl protein solution (30 mg/ml) and 1 µl of the precipitant solution were suspended over a reservoir containing the precipitant solution (1.6 M sodium citrate pH 6.5). Crystals used in data collection were transferred into Paratone-N oil and flash-cooled in a stream of liquid nitrogen at 110 K. Data sets were taken at SSRL beamline 9–2 and reduced using HKL2000 v 1.98.4
The initial phasing for the AtraPBP1-
Data Collection | ||
PDB code | 4INX | 4INW |
Ligand | Z11Z13-16OH | Z11Z13-16Ald |
Wavelength (Å) | 1.2000 | 1.1000 |
Resolution range (Å) | 50–1.85 (1.92–1.85) | 50–1.14 (1.18–1.14) |
Unique observations | 15,052 | 63,309 |
Total observations | 63,875 | 456,474 |
Completeness (%) | 98.3 (91.8) | 97.2 (91.4) |
Space group | ||
Rsym | 0.040 (0.113) | 0.058 (0.475) |
<I/σ(I)> | 28.4 (10.1) | 29.3 (2.6) |
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Resolution range (Å) | 34.10–1.85 | 28.7–1.14 |
Reflections used | 14,023 | 55,453 |
Rcryst (%) | 16.8 | 16.0 |
Rfree (%) | 21.4 | 18.3 |
# of protein, non-hydrogen atoms | 1,286 | 1,339 |
# of non-protein atoms | 177 | 208 |
rms bond length (Å) | 0.022 | 0.022 |
rms bond angles (°) | 1.87 | 1.80 |
Average main chain B values (Å2) | 13.01 | 18.18 |
Average ligand B values (Å2) | 30.33 | 33.67 |
The coordinates of Z11
Structural overlays were done using the programs SUPERPOSE and LSQKAB (CCP4 V.6.0.1 package) through CCP4i
The pH 6.5 AtraPBP1 structure complexed with either
AtraPBP1 (
The α-helices are sequentially labelled.
Structural overlay between the NMR structure at pH 4.5, the x-ray structure at pH 6.5 and the MD simulations to model the modifications at pH 7.0. Color code: blue (NMR structure at pH 4.5); light pink (x-ray structure at pH 6.5); yellow (pH 7.0 MD at t = 5 ps); orange (pH 7.0 MD at t = 10 ps); red (pH 7.0 MD at t = 12.5 ps). The pH-induced rotations observed in helices 1 and 7 are shown.
A) Structural overlay between the NMR structure at pH 4.5 (light red) and the x-ray structure at pH 6.5 (blue). The surface cavity at pH 6.5 is colored in blue and (11
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silkworm moth, |
1.1 | 67 | 137 | 171 | 1DQE |
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malaria mosquito, |
3.0 | 18 | 123 | 27 | 2ERB |
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cockroach, |
3.1 | 12 | 117 | 85 | 1ORG |
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honey bee, |
2.9 | 12 | 123 | 157 | 1TUJ |
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honey bee, |
2.8 | 15 | 117 | 128 | 3BJH |
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fruit fly, |
2.8 | 14 | 124 | 108 | 1OOH |
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Some OBPs are known to dimerize. In the cases of the mosquito proteins OBP1 from
The overall hook conformation of the hydrocarbon chain in both pheromone constituents is largely dictated by the
In the case of the
The alcoholic component,
Many odorant-binding proteins that have been studied are believed to load and release pheromones through a pH-dependent conformational rearrangement. This effect has first been described for BmorPBP, the PBP from the silkworm moth
In the present study, pheromone-bound crystal structures of AtraPBP1 at high pH bring complementary structural insights to the pH-dependent release mechanism for pheromones. At pH 6.5, the C-terminal helix α7 is partially disordered and extruded to accommodate the pheromone, which is consistent with the previous studies of AtraPBP1
Taken together, the analysis of the structures at pH 4.5 and 6.5 suggests a mechanism for ligand binding and dissociation. At acidic pH and in the absence of pheromone, the α7 helix rotates inside the hydrophobic binding pocket and is anchored by a pair of salt bridges (His80-Glu132, His95-Glu141). As the pH is increased from 4.5 to 7.0, the deprotonation of both His80 and His95 disrupts these interactions, enabling helix α7 to move outside of the binding cavity. Subsequently, helix α7 becomes disordered which contributes to opening a narrow path to the hydrophobic binding cavity, which is inadequate for the transit of the ligand (
A) MD at t = 0 ps; B) MD at t = 12.5 ps. The motion of the helix α7 is displayed in yellow. Electrostatic calculations are represented by blue for positive charge and red for negative charge with unit +5/−5 kT/e. Electrostatic calculations were done with APBS V1.2.1 and rendered with both VMD V1.8.7 and PyMOL V1.2
The mechanism by which AtraPBP1 load and unload pheromones involves synergistic movements of both helices α1 and α7 triggered while transitioning through a pH gradient. In addition, the protein's specificity for particular shapes and sizes of molecules may imply the existence of a filter which could be located at the entrance of the narrow tunnel leading to the inner binding cavity. Both the NMR structure at pH 4.5 and the x-ray structures at pH 6.5 are snapshots of the conformation of AtraPBP1 during the pH-induced structural transition. At pH 4.5, AtraPBP1 is devoid of ligand with the α7 helix occupying the inner binding cavity in a shut conformation inaccessible for the solvent. The transition from pH 4.5 to 6.5 results in significant conformational modifications with the synergetic movement of helices α1 and α7. However, the binding cavity at pH 6.5 is isolated from the solvent. Our data indicate that both
In order to understand the motions necessary to access the binding cavity, we used molecular dynamics (MD) to model the motions of the helices α1 and α7 that would occur in neutral pH environment. After a 12.5 ps (12,500 steps) MD simulation using the 1.1 Å refined crystal structure as a starting model, the helix α7 becomes fully disordered and rotates away exposing the long narrow channel to the hydrophobic pocket. Meanwhile, the α1 helix has a less significant movement and does not contribute to the channel opening. This agrees with the recent NMR analysis of AtraPBP1 at pH 7.0
In this study, we solved the high resolution structures of the pheromone-binding protein 1 from