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

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.