crystal-structure-n-terminal-domain-ryanodine-receptor-from-plutella

Crystal Structure of the N-terminal Domain of Ryanodine Receptor from <em>Plutella xylostella</em>

Development of potent and efficient insecticides targeting insect ryanodine receptors (RyRs) has been of great interest in the area of agricultural pest ...

Tianjin University

Fujian Agriculture and Forestry University

Ministry of Education, Fuzhou

Ryanodine receptors (RyRs) are large calcium-release channels located in sarcoplasmic reticulum membrane. They play a central role in excitation-contraction coupling of muscle cells. Three commercialized insecticides targeting pest RyRs generate worldwide sales over 2 billion U.S. dollars annually, but the structure of insect RyRs remains elusive, hindering our understanding of the mode of action of RyR-targeting insecticides and the development of insecticide resistance in pests. Here we present the crystal structure of RyR N-terminal domain (NTD) (residue 1-205) at 2.84 Å resolution from the diamondback moth (DBM), Plutella xylostella, a destructive pest devouring cruciferous crops all over the world. Similar to its mammalian homolog, DBM RyR NTD consists of a beta-trefoil folding motif and a flanking alpha helix. Interestingly, two regions in NTD interacting with neighboring domains showed distinguished conformations in DBM relative to mammalian RyRs. Using homology modeling and molecular dynamics simulation, we created a structural model of the N-terminal three domains, showing two unique binding pockets that could be targeted by potential species-specific insecticides. Thermal melt experiment showed that the stability of DBM RyR NTD was higher than mammalian RyRs, probably due to a stable intra-domain disulfide bond observed in the crystal structure. Previously DBM NTD was shown to be one of the two critical regions to interact with insecticide flubendiamide, but isothermal titration calorimetry experiments negated DBM NTD alone as a major binding site for flubendiamide.

Crystal structure of ryanodine receptor N-terminal domain from Plutella xylostella reveals two potential species-specific insecticide-targeting sites.

Calmodulin (CaM) is a Ca-binding protein that plays an important role in cellular Ca-signaling. CaM interacts with diverse downstream target proteins and regulates their functions in a Ca-dependent manner. CaM changes its conformation and hydrophobicity upon [Ca] change and consequently changes its interaction with CaM-binding domains from the targets. Based on these special properties of CaM, it was used as an affinity tag to develop a novel purification strategy by using it for two sequential orthogonal purification steps: 1) an affinity purification step, in which CaM-tag interacts with an immobilized CaM-binding domain; and 2) a hydrophobic interaction chromatography step, during which CaM binds to a phenyl sepharose column. In both steps, the CaM-tagged protein binds in the presence of Ca and unbinds in the presence of ethylenediaminetetraacetic acid (EDTA). An optional third step can be added to remove the CaM-tag if necessary. We used green fluorescent protein (GFP) as a test protein to demonstrate the effectiveness of the method. High yield and high purity of GFP with proper function was obtained using this novel strategy. We believe that this method can be applied to a wide range of protein targets for structural and functional studies.

Article	Year
Crystal structure of ryanodine receptor N-terminal domain from Plutella xylostella reveals two potential species-specific insecticide-targeting sites. Insect biochemistry and molecular biology\| PubMed ID: 29191465	2018
A two-step purification strategy using calmodulin as an affinity tag. Journal of chromatography. A\| PubMed ID: 29499842	2018

Lianyun Lin

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