Crystal Structure of the N-terminal Domain of Ryanodine Receptor from Plutella xylostella.

Development of potent and efficient insecticides targeting insect ryanodine receptors (RyRs) has been of great interest in the area of agricultural pest control. To date, several diamide insecticides targeting pest RyRs have been commercialized, which generate annual revenue of 2 billion U.S. dollars. But comprehension of the mode of action of RyR-targeting insecticides is limited by the lack of structural information regarding insect RyR. This in turn restricts understanding of the development of insecticide resistance in pests. The diamondback moth (DBM) is a devastating pest destroying cruciferous crops worldwide, which has also been reported to show resistance to diamide insecticides. Therefore, it is of great practical importance to develop novel insecticides targeting the DBM RyR, especially targeting a region different from the traditional diamide binding site. Here, we present a protocol to structurally characterize the N-terminal domain of RyR from DBM. The x-ray crystal structure was solved by molecular replacement at a resolution of 2.84 Å, which shows a beta-trefoil folding motif and a flanking alpha helix. This protocol can be adapted for the expression, purification and structural characterization of other domains or proteins in general.

A two-step purification strategy using calmodulin as an affinity tag.

Calmodulin (CaM) is a Ca2+-binding protein that plays an important role in cellular Ca2+-signaling. CaM interacts with diverse downstream target proteins and regulates their functions in a Ca2+-dependent manner. CaM changes its conformation and hydrophobicity upon [Ca2+] 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 Ca2+ 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.