Crystal structure of diamondback moth ryanodine receptor Repeat34 domain reveals insect-specific phosphorylation sites

Abstract

Background Ryanodine receptor (RyR), a calcium-release channel located in the sarcoplasmic reticulum membrane of muscles, is the target of insecticides used against a wide range of agricultural pests. Mammalian RyRs have been shown to be under the regulatory control of several kinases and phosphatases, but little is known about the regulation of insect RyRs by phosphorylation. Results Here we present the crystal structures of wild-type and phospho-mimetic RyR Repeat34 domain containing PKA phosphorylation sites from diamondback moth (DBM), a major lepidopteran pest of cruciferous vegetables. The structure has unique features, not seen in mammalian RyRs, including an additional α-helix near the phosphorylation loop. Using tandem mass spectrometry, we identify several PKA sites clustering in the phosphorylation loop and the newly identified α-helix. Bioinformatics analysis shows that this α-helix is only present in Lepidoptera, suggesting an insect-specific regulation. Interestingly, the specific phosphorylation pattern is temperature-dependent. The thermal stability of the DBM Repeat34 domain is significantly lower than that of the analogous domain in the three mammalian RyR isoforms, indicating a more dynamic domain structure that can be partially unfolded to facilitate the temperature-dependent phosphorylation. Docking the structure into the cryo-electron microscopy model of full-length RyR reveals that the interface between the Repeat34 and neighboring HD1 domain is more conserved than that of the phosphorylation loop region that might be involved in the interaction with SPRY3 domain. We also identify an insect-specific glycerol-binding pocket that could be potentially targeted by novel insecticides to fight the current resistance crisis. Conclusions The crystal structures of the DBM Repeat34 domain reveals insect-specific temperature-dependent phosphorylation sites that may regulate insect ryanodine receptor function. It also reveals insect-specific structural features and a potential ligand-binding site that could be targeted in an effort to develop green pesticides with high species-specificity.

Regulation of GSTu1-mediated insecticide resistance in Plutella xylostella by miRNA and lncRNA

“…xylostella (L.), a major pest of cruciferous crops, is one of the most resistant insects globally [6,7]. Chlorantraniliprole is an anthranilic diamide insecticide that shows remarkable efficacy in the control of several orders of insect pests, especially lepidopteran pests, but has low toxicity towards untargeted organisms [8,9]. However, in recent years, P. xylostella has developed high-level resistance to chlorantraniliprole in many countries [10][11][12].…”

The Role of Insect Cytochrome P450s in Mediating Insecticide Resistance

“…However, there are not many reports on insect phosphorylation-mediated resistance. One example is known for regulation of acetylcholine receptor (nAChR) signaling, where resistance to imidacloprid is regulated by intracellular phosphorylation [119]; P. xylostella-specific PKA phosphorylation sites may regulate insect ryanodine receptor function [120]. There are limited cases of P450 regulation.…”

Review on Structures of Pesticide Targets

“…Some new potent insecticide targets have been discovered. The crystal structure of the RyR’s phosphorylation [ 45 ] and N-terminal [ 46 ] domains, as well as the SPRY2 domain from Plutella xylostella [ 47 ], were solved. These structures provide insights into the development of novel insecticides [ 48 ].…”