果蝇钙调蛋白和肌球蛋白NINAC相互作用分析

果蝇的视觉信号转导途径是已知的最快的G 蛋白偶联信号通路。这其中涉及到TRP/TRPL通道的开放以及钙离子的内流等一系列反应的形成。NINAC（neither inactivation nor afterpotential C）是一种特异性存在于果蝇感光细胞中的第3类肌球蛋白（Myosin III），其在终止果蝇的视觉信号转导通路中起着非常重要的作用。NINAC蛋白具有两种亚型：一种是132 kD的蛋白亚型 (p132)，另一种则是174 kD的蛋白亚型(p174)。这两种不同的蛋白亚型都具有相同的激酶催化结构域(kinase domain)，以及与肌球蛋白相似的马达结构域(motor domain)。但是，它们在C末端却存在着非常大的差异，这其中包括了钙调蛋白结合基序(IQ motif)。NINAC的这两种蛋白亚型在果蝇的感光细胞中的定位以及作用有很大不同，尤其是在与钙调蛋白的相互作用方面。钙调蛋白结合基序与钙调蛋白（CaM）之间的相互作用对于果蝇的视觉信号通路具有重要的意义：NINAC结合钙调蛋白能力的缺失将导致果蝇的视觉传导缺陷。本文通过蛋白共表达的方法，成功表达并纯化得到了不同版本的NINAC与钙调蛋白的蛋白复合物。静态光散射的结果表明，在Ca2+存在情况下，p174蛋白可以结合2个Ca2+-CaM，而p132只结合1个Ca2+-CaM。通过分析型凝胶过滤以及等温量热滴定技术，进一步鉴定了p174及p132的IQ2（第2个钙调蛋白结合基序）序列与Ca2+ CaM的相互作用。通过序列分析及进一步的突变实验发现，p174 IQ2中的3个疏水氨基酸（F1083，F1086 和 L1092）对于钙调蛋白的结合非常重要，并导致了p174与p132蛋白和Ca2+ CaM结合能力的差异。本文的研究提供了NINAC与Ca2+-CaM相互作用的生化机制，将为进一步在果蝇视觉信号通路中深入研究CaM是如何调节NINAC的体内功能实验打下基础。

Characterization of the Interaction between Ca 2+-CaM and NINAC

The Drosophila visual cascade is the fastest known G-protein-coupled signaling pathway involving the opening of TRP/TRPL channels and the influx of Ca2+ ion. The NINAC (neither inactivation nor afterpotential C) is a member of class III myosins, which specifically localizes to the photoreceptor cell and plays a significant role in the termination of the Drosophila visual transduction. There are two isoforms of NINAC: the p132 and the p174, which share a protein kinase catalytic domain (kinase domian), a motor domain, but differ in their C terminal region which contains calmodulin binding domains (IQ motif). The two isoforms have remarkable differences in the photoreceptor localization and the calmodulin (CaM) binding capability. The interactions between the IQ motifs and the CaM are very important for the Drosophila visual signaling pathway: the mutations disrupting the interactions between the NINAC and the CaM will lead to defects in the visual transduction. In this paper, we successfully co expressed and purified different versions of the NINAC/Ca2+-CaM protein complexes in vitro. Using the size exclusion chromatography coupled with the multi-angle laser light scattering (SEC-MALS) method, we found that the p174 could bind to two Ca2+-CaMs, whereas the p132 could only bind to one Ca2+-CaM. Utilizing the analytical gel filtration and isothermal titration calorimetry (ITC) techniques, we further characterized the interactions between the p174/p132 IQ2 motifs (the second calmodulin binding motif) and the Ca2+-CaM. Combined with the sequence analysis and the following mutagenesis experiments, we found that three hydrophobic amino acids (F1083, F1086 and L1092) in the p174 IQ2 motif were critical for the Ca2+-CaM binding and caused the Ca2+-CaM binding differences between the p174 and the p132. In summary, our findings provide further understanding of the biochemical mechanisms of the NINAC/Ca2+ CaM interaction and will contribute to the future in vivo functional studies of NINAC’s regulation by CaM in the Drosophila visual transduction.