Abstract
Insect neuropeptide receptors are among the potential targets for designing next-generation pesticides. Activation of allatostatin receptor type C (AstR-C), a G Protein-coupled receptor (GPCR), upon stimulation with its endogenous ligand, allatostatin C (AST-C), leads to the inhibition of juvenile hormone (JH) secretion that consequently regulates physiology of insects. Here we conducted in silico and in vitro approaches to characterize the structure and function of AstR-C of Thaumetopoea pityocampa (T.pit), a well-known pest in Mediterranean countries. The sequence of AstR-C and AST-C were derived from whole genome sequencing (WGS) data. Resonance energy transfer (RET) methods were used to investigate the downstream effectors of the receptor and the temporal kinetics of G protein activation. Three-dimensional (3D) structure of AstR-C constructed via homology modeling methods was subjected to molecular dynamics (MD) simulations and docking studies to identify the orthosteric pocket. Our results showed that T.pit AstR-C couples to Gαi/o subtype of G proteins at sub-nanomolar ranges of the the ligand with the G protein recruitment and activation kinetics of ∼4 and 6 seconds, respectively, when 1 nM AST-C is administered. At the increasing concentration of native ligand, βarrestin was shown to be recruited at nanomolar ranges the ligand. Docking and MD simulation studies revealed the importance of extracellular loop 2 (ECL2) in T.pit AstRC/AST-C interaction, and combination of in silico and in vitro methods supported the accuracy of the built model and the predicted orthosteric pocket. Q2716.55 (Ballesteros-Weinstein generic numbering) was found to have a substantial role in G protein dependent activation of AstR-C possibly via contributing to the flexibility of the structure.
Competing Interest Statement
The authors have declared no competing interest.
Abbreviations
- AstR-C
- allatostatin receptor type C
- GPCRs
- G protein-coupled receptors
- AST-C
- allatostatin C
- JH
- Juvenile hormone
- T.pit
- Thaumetopoea pityocampa
- WGS
- whole genome sequencing
- RET
- Resonance energy transfer
- 3D
- Three-dimensional
- MD
- molecular dynamics
- ECL
- extracellular loop
- ICL
- intracellular loop
- FRET
- Förster resonance energy transfer
- BRET
- Bioluminescence resonance energy transfer
- YFP
- yellow fluorescent protein
- CFP
- cyan fluorescent protein
- Cα
- Carbon alpha
- RMSD
- root mean square deviations
- RMSF
- root mean square fluctuations
- Ala
- Alanine
- DCCM
- dynamical cross-correlation map
- PDB
- protein data bank
- BSA
- bovine serum albumin
- PBS
- phosphate buffered saline
- MM/GBSA
- Molecular Mechanics/ Generalized Born Surface Area.