Structural basis for diamide modulation of ryanodine receptor

Abstract

Diamide insecticides target insect ryanodine receptors (RYRs) and cause dysregulation of calcium signaling in insect muscles and neurons, generating worldwide sales over 2 billion US dollars annually. Several resistance mutations have been reported to reduce the efficacy of the diamides, but the exact binding sites and mechanism of resistance mutations were not clear. Recently, we solved the cryo-electron microscopy (cryo-EM) structure of RYR in complex with the anthranilic diamide chlorantraniliprole (CHL). CHL binds to the pseudo–voltage-sensor domain (pVSD) of RYR, a site in proximity to the previously identified resistance mutations. Mutagenesis studies in silico, in mutant cell lines, and in transgenic Drosophila strains revealed the key residues involved in diamide coordination and the molecular mechanism under species-selectivity and resistance mutations. We also proposed that CHL may alleviate the loss-of-function effects of some central core disease (CCD) mutations by increasing the opening probability (Po) of RYR1. In addition, we solved the crystal structures of several RYR domains from the diamondback moth and the bee, revealing insect-specific structural features which could be potentially targeted by novel insecticides. Interestingly, we found that the phosphorylation of insect RYR is temperature dependent, facilitated by the low thermal stability and dynamic structure of the insect RYR. Our structures provide a foundation for developing novel pesticides to overcome the resistance crisis.

First Report of Helicoverpa armigera (Lepidoptera: Noctuidae) Resistance to Flubendiamide in Brazil: Genetic Basis and Mechanisms of the Resistance

“…The partial sequencing of the ryanodine receptor genes of H. armigera-resistant strains to flubendiamide showed no mutations in the region that covers the transmembrane regions II to VI. This C-terminal region was formerly described to contain target site mutations [15,47]. The primary lepidopteran resistance mechanism to diamides is the mutation in RyR, which can promote very high resistance ratios [15].…”

Calmodulin Modulation of Insect Ryanodine Receptor

“…Of these, CaMBD2 and CaMBD3 showed nanomolar a nity to Ca 2+ -CaM in both RyR1 and RyR2 at the high [Ca 2+ ] cyto condition and micromolar a nity to apo-CaM at the low [Ca 2+ ] cyto condition, suggesting that these sites are more likely to be the CaM-binding regions in RyR. Recently, several cryo-EM structures of full-length RyRs in complex with CaM were solved, con rming that CaMBD2 is the genuine CaM-binding domain in both RyR1 and RyR2 (Gong et al, 2019;Ma et al, 2020;Maximciuc et al, 2006). Under low [Ca 2+ ] cyto condition, the C-lobe of apo-CaM binds to the surface of CaMBD2 from the central domain and another -helix from the handle domain, whereas the N-lobe interacts with multiple -helices from the HD1 domain (Gong et al, 2019;Ma et al, 2020;Maximciuc et al, 2006).…”

“…The diamide insecticide family, such as ubendiamide (FLU), chlorantraniliprole (CHL), and cyantraniliprole (CYA), can speci cally target a range of agricultural pests, including lepidopteran, hemipteran, and coleopteran pests by activating their RyRs (Cordova et al, 2006;Tohnishi et al, 2005). A recent study by cryo-electron microscopy (EM) revealed that the binding site of diamide insecticide is located at the pseudo voltage-sensor domain (pVSD) in the transmembrane region of RyR (Ma et al, 2020). Interestingly, four mutations associated with diamide-resistance, G4946E, I4790M, Y4701D and Y4922F (diamondback moth numbering), are located exactly at the identi ed diamide binding site and drastically lower their e cacy by hundreds of folds (Ma et al, 2020).…”

“…A recent study by cryo-electron microscopy (EM) revealed that the binding site of diamide insecticide is located at the pseudo voltage-sensor domain (pVSD) in the transmembrane region of RyR (Ma et al, 2020). Interestingly, four mutations associated with diamide-resistance, G4946E, I4790M, Y4701D and Y4922F (diamondback moth numbering), are located exactly at the identi ed diamide binding site and drastically lower their e cacy by hundreds of folds (Ma et al, 2020). As a giant modular protein with a molecular weight over 2 megadaltons, the activity of RyR is regulated by many modulatory proteins and small molecules, including FK506-binding protein (FKBP), calmodulin (CaM), ATP, caffeine, calcium, PCB95, ryanodine, and diamides (Balshaw et al, 2001;Marx et al, 1998;Porta et al, 2012;Van Petegem, 2015).…”

“…Recently, several cryo-EM structures of full-length RyRs in complex with CaM were solved, con rming that CaMBD2 is the genuine CaM-binding domain in both RyR1 and RyR2 (Gong et al, 2019;Ma et al, 2020;Maximciuc et al, 2006). Under low [Ca 2+ ] cyto condition, the C-lobe of apo-CaM binds to the surface of CaMBD2 from the central domain and another -helix from the handle domain, whereas the N-lobe interacts with multiple -helices from the HD1 domain (Gong et al, 2019;Ma et al, 2020;Maximciuc et al, 2006). In contrast, under high [Ca 2+ ] cyto condition, both lobes of Ca 2+ -CaM wrap around CaMBD2 and have extensive interactions (Gong et al, 2019;Ma et al, 2020;Maximciuc et al, 2006), which are consistent with the previous ITC experiments (Lau et al, 2014).…”