Identification and characterization of a novel calmodulin binding site in Drosophila TRP C-terminus
Highlights: • Drosophila TRP 783–862 is a novel calmodulin binding site. • The shortest Ca{sup 2+}-CaM interaction region and core CaM binding sequences in TRP 783–862 were dissected. • Ca{sup 2+}-CaM binding induces helical structure formation of Drosophila TRP 783–862. Transient receptor potential (TRP) channels are a group of essential cation channels involved in many important sensory signal transduction processes, such as light, temperature, tastes and pressure sensing. Drosophila TRP channel is the first discovered family member and plays important roles in photo-transduction in Drosophila. Calmodulin (CaM), an important downstream effector of Ca{sup 2+} signal, was considered as a vital regulator of TRP activities. In this study, we discovered a novel Ca{sup 2+} dependent CaM binding site (TRP 783–862) in between the previously reported two calmodulin binding sites (CBSs). The isothermal titration calorimetry (ITC) and the size exclusion chromatography coupled with multi-angle static light scattering (SEC-MALS) results showed that the dissociation constant (Kd) between TRP 783–862 and Ca{sup 2+}-CaM is 0.10 ± 0.04 μM and their binding stoichiometry is 1:1. In addition, the shortest Ca{sup 2+}-CaM interaction region and core CaM binding sequences in TRP 783–862 were dissected by the boundary mapping and mutagenesis experiments. More interestingly, by comparing the circular dichroism (CD) spectra before and after Ca{sup 2+}-CaM binding, the TRP 783–862 fragment showed Ca{sup 2+}-CaM binding dependent secondary structure changes, indicating that the interaction between CaM and Drosophila TRP channel may have a conformational impact on TRP structure. In summary, by identifying and characterizing a novel CaM binding site in TRP C-terminus, our findings provided a biochemical and structural basis for further in vivo functional studies of Ca{sup 2+}-mediated TRP channel regulation through CaM/TRP interaction.