Identification and characterization of a novel calmodulin binding site in Drosophila TRP C-terminus
Identification and characterization of a novel calmodulin binding site in Drosophila TRP C-terminus
复制标题
果蝇 TRP C 末端新型钙调蛋白结合位点的鉴定和表征
DOI：
10.1016/j.bbrc.2018.05.007
复制
发表时间：
2018
影响因子：
3.1
通讯作者：
Liu Wei
中科院分区：
文献类型：
--
作者：
Sun ZiLing;Zheng YunHua;Liu Wei
瞬时受体电位（transmittance receptor potential，TRP）通道是一组重要的阳离子通道，参与光、温度、味觉和压力等多种重要的感觉信号转导过程，果蝇TRP通道是最早发现的家族成员，在果蝇的光信号转导中起重要作用。钙调素（CaM）是Ca ~（2+）信号的重要下游效应子，被认为是TRP活性的重要调节因子。在本研究中，我们发现了一个新的钙离子依赖的钙调素结合位点（TRP 783-862）之间的两个钙调素结合位点（CBS）。等温滴定量热法（ITC）和体积排阻色谱-多角度静态光散射（SEC-MALS）结果表明，TRP 783-862与Ca 2 +-CaM的解离常数（Kd）为0.10 ± 0.04 μM，其结合化学计量比为1：1。此外，通过边界作图和突变实验，对TRP 783-862中最短的Ca ~（2+）-CaM相互作用区域和核心CaM结合序列进行了解剖。通过比较Ca 2 +-CaM结合前后TRP 783-862片段的圆二色性（CD）谱，发现TRP 783-862片段的二级结构发生了依赖于Ca 2 +-CaM结合的变化，表明CaM与果蝇TRP通道之间的相互作用可能对TRP结构产生了构象影响。总之，通过鉴定和表征TRP C-末端的新的CaM结合位点，我们的研究结果提供了进一步的生物化学和结构基础，通过CaM/TRP相互作用的Ca 2+介导的TRP通道调节的体内功能研究。
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.DrosophilaTRP channel is the first discovered family member and plays important roles in photo-transduction inDrosophila. Calmodulin (CaM), an important downstream effector of Ca2+signal, was considered as a vital regulator of TRP activities. In this study, we discovered a novel Ca2+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 Ca2+-CaM is 0.10 ± 0.04 μM and their binding stoichiometry is 1:1. In addition, the shortest Ca2+-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 Ca2+-CaM binding, the TRP 783–862 fragment showed Ca2+-CaM binding dependent secondary structure changes, indicating that the interaction between CaM andDrosophilaTRP 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 furtherin vivofunctional studies of Ca2+-mediated TRP channel regulation through CaM/TRP interaction.