Structural elucidation of ETHR-A and ETHR-B from Plutella xylostella and insight into non-conservative mutations in transmembrane helix-6.

The development of Diamondback moth (DBM) depends on the ecdysis triggering hormone receptor (ETHR); a neuronal membrane G-protein coupled receptor (GPCR) connected to the metamorphosis cascade. Lepidopteran insect DBM is an infamous pest of cruciferous plants. This study examined the full-length coding sequences (CDS) of PxETHR-A and PxETHR-B from the DBM genome. The three-dimensional (3 D) models of both receptors were generated in an inactive state. The behaviour and stability of receptors were examined using molecular dynamics simulations in a lipid membrane system for 300 ns and established a GPCR family-based view. Secondary interactions within receptors were studied to know more about factors contributing to their stability. Multiple sequence alignment revealed conserved features of insect ETHRs those compared to the GPCR family proteins. These features were helpful during the evaluation of the molecular models of both receptors. Side-chain orientation of conserved residues, non-conserved and conserved hydrogen-bond networks (HBN) and hydrophobic clusters were examined in the structures of both receptors. The non-conserved residues L6.35, T6.39, C/S6.43, and L6.48, are present in a conserved position on the transmembrane helix-6 (TM6) of ETHRs. In TM6, PxETHR-A and PxETHR-B differ at positions C/S6.43 and Y/F6.51, both being part of the HBN.Communicated by Ramaswamy H. Sarma.

In silico Binding Profile Analysis and In vitro Investigation on Chitin Synthase Substrate and Inhibitors from Maize Stem Borer, Chilo partellus.

INTRODUCTION: Insect growth and metamorphosis are strictly dependent on the structural changes that occur in chitin containing tissues and organs. Chitin synthase catalyzes chitin polymerization by ß-(1, 4) glycosidic linkage of N-acetyl-D-glucosamine (GlcNAc) monomers; the major component of insect cuticles. Targeting this enzyme could be a promising strategy to control insect pests while avoiding adverse effects on coexisting populations. Nikkomycin Z and polyoxins are commercially available fungal inhibitors known to bind to the nucleotide-binding sites of insects and fungal chitin synthase. But the binding mode of chitin synthase has not been explored to date as its structure is not available yet. METHODS: To understand the structural features of the Chilo partellus chitin synthase enzyme (CpCHS), the three-dimensional (3D) structure of the CpCHS catalytic domain was modeled using ROBETTA webserver. The obtained model was used to investigate the binding mode of its substrate, uridine diphosphate-N-acetyl-D-glucosamine (UDP-GlcNAc), and inhibitors (nikkomycin Z and polyoxins) by molecular docking approach using Schrödinger Suite-Maestro v9.2. The docked complexes were further investigated for their interaction stability by performing molecular dynamics (MD) simulations using GROMACS v5.1.2. RESULTS: Our study highlighted the significance of various interactions made by CHS residues present in the Walker-B loop and donor-binding motifs with the substrate (UDP-GlcNAc), and GEDR motif with an acceptor (GlcNAc). Also, the interactions of the QRRRW motif while forming chitin polymer were explored. We observed that the inhibitors exhibited good binding affinity with these motifs, indicated by their docking and binding affinity scores. CONCLUSION: In vitro analysis suggested that nikkomycin Z showed higher inhibition of chitin synthase activity at a concentration of 2.5 µg.L-1. Our study provided insights into the crucial interactions of chitin synthase while designing inhibitors against insect pests.