Mechanistic insight into binding interaction between chemosensory protein 4 and volatile larval pheromones in honeybees (Apis mellifera)

https://doi.org/10.1016/j.ijbiomac.2019.09.041Get rights and content

Abstract

Honeybees communicate with members of their intra-species via pheromones. The volatile pheromones, β-ocimene and allo-ocimene, are the primary signals of larvae to beg for the care from the nurses. Of the odorant binding proteins (OBPs)/chemosensory proteins (CSPs), CSP4 has the best affinity with β-ocimene and allo-ocimene. To reveal the binding mechanism of CPS4 with them, fluorescent quenching, UV absorption spectra, circular dichroism (CD) spectra, isothermal titration calorimetry (ITC), molecular docking, molecular dynamic (MD) simulation, and site-directed mutagenesis were applied. The quenching constant Ksv decreased with temperature increase, and the interaction distance was 2.73 nm and 2.43 nm (<10 nm), indicating that β-ocimene and allo-ocimene could form stable complexes with CSP4. The observed △H < 0 and △S > 0 of thermodynamics suggest the main driving forces are electrostatic or hydrophobic force. All above thermodynamics findings are in line with the results of ITC experiments. Furthermore, molecular docking, MD simulation and site-directed mutagenesis indicate the binding cavities are located at cavity 1 in C-terminal of CSP4, where Tyr98 and Asp67 are vital amino acids in maintaining the stable form of protein and larval pheromones, and electrostatic energies are the main driving forces. Our findings gain novel insight into the binding mechanism of chemosensory protein with volatile larval pheromones and are important for understanding olfactory interaction of honeybees.

Introduction

Sense of smell enables insects to locate mates, food sources, and oviposition sites. For social insects, the olfactory system of honeybees (Apis mellifera) is critical for detection and discrimination of olfactory cues, which is the predominant mode of coordination in the societies and is essential for colony communication [1,2]. The olfactory system is orchestrated at various levels, starting with reception of odor at the periphery, processing of signals at the antennal lobes and the higher centers of the brain, and ultimately translation of olfactory signals into behavior and physiological changes [3]. Two families of olfaction-related small soluble proteins, odorant binding proteins (OBPs) and chemosensory proteins (CSPs) act as carriers for hydrophobic odors or pheromones through the aqueous sensillar lymph as the first step of signal transduction [4]. In the genome of A. mellifera, 21 OBPs and 6 CSPs are identified [5,6]. All these olfactory proteins can transport a variety of odor molecules and pheromones to odorant receptors in insects [4]. Therefore, it is necessary to study the binding function of OBPs and CSPs.

The typical characteristics of OBPs and CSPs in insects are the internal hydrophobic cavity that is favorable to bind suitable ligands [7]. To date, only 20 OBPs and 3 CSPs structures have been characterized in insects [8]. The methods for analyzing structure in common are X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy. The first reported structure of OBP is the pheromone binding protein (PBP) of Bombyx Mori [9], and the first CSP structure is CSPMbraA6 of the moth Mamestra brassicae [10]. In honeybees, the structures of OBP1 and OBP14 are established by X-ray diffraction, of which the structure of OBP1 could be altered at pH 4.0, 5.5, and 7.0 [11]. For OBP14, the cavity is quite variable and can reshape to bind different odorant molecules [12]. These findings are suggestive of the fact that the pH of solutions and ligands could impact on the structure of binding proteins in honeybees.

Comparing to structure analysis, fluorescence assay is an efficient platform to study the binding activity of OBPs or CSPs, which provide essential information for understanding their physiological function [13]. OBP1 (Antenna-specific protein 1, ASP1) is selectively expressed in workers and drones with a good affinity for mandibular pheromones of queen bees, which could have an impact on social behavior and physiology of bees [14,15]. OBP2 (Antenna-specific protein 2, ASP2) is able to bind 2-heptanone [16], an alarm pheromone that could mark bitten individuals [17]. OBP3, highly expressed in mated queen bees, could bind with benzoate [18,19]. The gene of Obp5 is highly expressed in 10- and 15-day-old workers and has a good affinity with both benzyl alcohol and 2-phenylethanol, which are present in the volatile compounds of chalkbrood disease-infected larvae [20]. OBP13 is mostly abundant in young worker larvae and virgin queens, and specifically binds with oleic acid and some structurally related compounds [21] which are emitted by the brood and cause long-term alteration to the receiver's physiological [22,23]. OBP14 is highly abundant in the mandibular glands of hive bees and better associated with monoterpenoid structures [21], which improve the olfactory learning and memory [24]. OBP16 and OBP18 have a strong binding force to β-ocimene and oleic acid, respectively, which are associated with hygienic behavior [25]. OBP21 is abundant in old bees and binds farnesol [21], a trail pheromone that attracts honeybee swarms [26]. However, olfactory functions of CSPs in honeybees are poorly understood in the peripheral chemosensory system. CSP3 (Antenna-specific protein 3, ASP3) is shown to bind specifically to large fatty acids and ester derivatives [27] that is emitted by the brood, and thus affect the behavior of nurse bees [23]. All these evidences are indicative of the fact that OBPs and CSPs are able to bind with a wide range of ligands, but are inclined to transport different odors or honeybee pheromones with a high binding force.

As a social insect, the cost of reproduction and brood care lead to central trade-offs in life-history. In honeybee colonies, all brood care is performed by the nurse bees [28]. To prompt identify and accept the larvae in the comb cells, efficient interactions between larvae and worker bees are required. There are two classes of brood pheromones: non-volatile 10 fatty aliphatic esters and volatile β-ocimene [29,30]. The 10 fatty aliphatic esters urge the adults to feed larvae and cap the brood cells [23,31]. Starving honeybee larvae evolve a skill to beg the need for food from nurse bees by releasing β-ocimene, a pheromone signal to which worker bees react by attending brood [32]. Additional, our recent works have identified a new volatile larval pheromone allo-ocimene, which could also elicit antennal signaling and attract nurse bees to care for the larvae (In press). However, there is no more relevant research of combine mechanism or process between binding proteins and volatile larval pheromones. To this end, we used a fluorescence competitive binding experiment to find the strongest binding force between the olfactory proteins and volatile larval pheromones. Then, we applied fluorescence quenching assays, circular dichroism (CD) spectra, isothermal titration calorimetry (ITC), and UV-absorption spectra to reveal the physicochemical mechanism of how volatile larval pheromones are carried by the transporter through the hydrophilic lymph of specialized sensilla. Furthermore, homology modeling, molecular docking and dynamic simulation, and site-directed mutagenesis were employed to explore the binding mode, and to confirm the key amino acids and their function during the binding processes of the protein with volatile larval pheromones.

Section snippets

Materials and methods

The recombinant plasmid pET-30a (+)-OBPs/CSPs were constructed and kept in our laboratory. The kits used for PCR and plasmid extraction were bought from TaKaRa (Japan). Primers were synthesized by Sangon biotech Co. Ltd. (Shanghai, China). Protein Ni-NTA Resin kit and the site directed mutagenesis system were purchased from Transgen Biotech Co. Ltd. (Beijing, China). The reagents of protein electrophoresis were purchased from Beyotime Biotechnology (Shanghai, China). The β-ocimene (purity >99%)

OBPs and CSPs exhibit varied binding affinities to volatile larval pheromones

To test the most relevant of OBPs and CSPs interacted with larval pheromones, the binding force between these proteins with β-ocimene and allo-ocimene were compared in a competitive fluorescence binding assay. Firstly, all of the OBPs and CSPs tested had good binding affinities with fluorescence reporter 1-NPN. With increasing concentrations of 1-NPN, the binding between 1-NPN and OBPs or CSPs were gradually saturated. Secondly, varied affinity OBPs/CSPs bound with β-ocimene and allo-ocimene

Discussion

The complex chemicals produced by the honeybee larvae are necessary to induce the behavior and the physiology of workers to care for the needs of the brood. OBPs and CSPs are the odor molecular carriers that transport larval pheromones through the sensillar lymph to ORs. Of all tested OBPs and CSPs, CSP4 is the olfactory protein with the strongest binding affinity to allo-ocimene. To explore the interaction mechanism of CSP4 with volatile larval pheromones, β-ocimene and allo-ocimene, a wide

Conclusion

Our data manifest the mechanism by characterization of the interaction between CSP4 and volatile larval pheromones, β-ocimene and allo-ocimene. Both of the two ligands could form stable complexes with CSP4, which are crucial for delivering the both pheromones through the sensillar lymph. Moreover, the binding sites located at the external cavity of CSP4 that could rapid transmit of the pheromones. The efficient transport and immediate release the pheromones are beneficial for worker bees to

Declaration of competing interest

The authors declare that they have no conflicts of interest with the contents of this article.

Acknowledgments

This work was supported by the Agricultural Science and Technology Innovation Program (CAAS-ASTIP-2015-IAR), the Earmarked Fund for Modern Agro-Industry Technology Research System (CARS-45), National Natural Science Foundation of China (No. 31601169) and National project for upgrading beekeeping industry of China. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Author contributions

FW, L-S Y and J-K L: conceived and designed the experiments; FW, Y-L F and BH: performed the experiments; HH, L-F M, MF and CM: analyzed the data; FW and J-K L: drafted and revised the manuscript.

References (54)

  • Y.J. Hu et al.

    Studies of interaction between colchicine and bovine serum albumin by fluorescence quenching method

    J. Mol. Struct.

    (2005)
  • C. Weng et al.

    Binding interaction between a queen pheromone component HOB and pheromone binding protein ASP1 of Apis cerana

    Int. J. Biol. Macromol.

    (2015)
  • Y. Sun et al.

    Study of interaction between protein and main active components in Citrus aurantium L. by optical spectroscopy

    J. Lumin.

    (2010)
  • D.C. Gilley et al.

    Volatile compounds emitted by live European honey bee (Apis mellifera L.) queens

    J. Insect Physiol.

    (2006)
  • J.D. Laughlin et al.

    Activation of pheromone-sensitive neurons is mediated by conformational activation of pheromone-binding protein

    Cell

    (2008)
  • C.G. Dosremedios et al.

    Fluorescence resonance energy-transfer spectroscopy is a reliable ruler for measuring structural-changes in proteins-dispelling the problem of the unknown orientation factor

    J. Struct. Biol.

    (1995)
  • J. Nardella et al.

    Endogenous fatty acids in olfactory hairs influence pheromone binding protein structure and function in Lymantria dispar

    Arch. Biochem. Biophys.

    (2015)
  • F.J. Richard et al.

    Intracolony chemical communication in social insects

    Insect. Soc.

    (2013)
  • M. Schott et al.

    Insect antenna-based biosensors for in situ detection of volatiles

    Adv. Biochem. Eng. Biotechnol.

    (2013)
  • W.S. Leal

    Odorant reception in insects: roles of receptors, binding proteins, and degrading enzymes

    Annu. Rev. Entomol.

    (2013)
  • P. Pelosi et al.

    Soluble proteins in insect chemical communication

    Cell. Mol. Life Sci.

    (2006)
  • S. Foret et al.

    Function and evolution of a gene family encoding odorant binding-like proteins in a social insect, the honey bee (Apis mellifera)

    Genome Res.

    (2006)
  • V. Campanacci et al.

    Moth chernosensory protein exhibits drastic conformational changes and cooperativity on ligand binding

    Proc. Natl. Acad. Sci. U. S. A.

    (2003)
  • E. Danty et al.

    Cloning and expression of a queen pheromone-binding protein in the honeybee: an olfactory-specific, developmentally regulated protein

    J. Neurosci.

    (1999)
  • L. Briand et al.

    Disulfide pairing and secondary structure of ASP(1), an olfactory-binding protein from honeybee (Apis mellifera L)

    J. Pept. Res.

    (2001)
  • L. Briand et al.

    Ligand binding and physico-chemical properties of ASP2, a recombinant odorant-binding protein from honeybee (Apis mellifera L.)

    Eur. J. Biochem.

    (2001)
  • F.R. Dani et al.

    Mapping the expression of soluble olfactory proteins in the honeybee

    J. Proteome Res.

    (2010)
  • Cited by (12)

    • Differences in ASP1 expression and binding dynamics to queen mandibular pheromone HOB between Apis mellifera and Apis cerana workers reveal olfactory adaptation to colony organization

      2022, International Journal of Biological Macromolecules
      Citation Excerpt :

      The components in QMP are relatively non-volatile; thus, their spread in the colony is largely dependent on the direct contact between attendant bees and other members [29]. OBPs and CSPs are compatible with many odors or pheromones; however, they could predispose the transport of certain molecules with high binding forces to improve the efficiency of performing specific tasks [16,30]. Our competitive binding assays showed that the HOB-binding force of Am-ASP1 was less affected by the increase of temperature compared with that of Ac-ASP1.

    View all citing articles on Scopus
    View full text