Wafer Lucas N, Tzul Franco O, Pandharipande Pranav P, McCallum Scott A, Makhatadze George I
Department of Biological Sciences, Rensselaer Polytechnic Institute, Troy, New York, 12180; Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, New York, 12180.
Protein Sci. 2014 Sep;23(9):1247-61. doi: 10.1002/pro.2506. Epub 2014 Jul 2.
Calmodulin (CaM) is a multifunctional messenger protein that activates a wide variety of signaling pathways in eukaryotic cells in a calcium-dependent manner. CaM has been proposed to be functionally distinct from the S100 proteins, a related family of eukaryotic calcium-binding proteins. Previously, it was demonstrated that peptides derived from the actin-capping protein, TRTK12, and the tumor-suppressor protein, p53, interact with multiple members of the S100 proteins. To test the specificity of these peptides, they were screened using isothermal titration calorimetry against 16 members of the human S100 protein family, as well as CaM, which served as a negative control. Interestingly, both the TRTK12 and p53 peptides were found to interact with CaM. These interactions were further confirmed by both fluorescence and nuclear magnetic resonance spectroscopies. These peptides have distinct sequences from the known CaM target sequences. The TRTK12 peptide was found to independently interact with both CaM domains and bind with a stoichiometry of 2:1 and dissociations constants Kd,C-term = 2 ± 1 µM and Kd,N-term = 14 ± 1 µM. In contrast, the p53 peptide was found to interact only with the C-terminal domain of CaM, Kd,C-term = 2 ± 1 µM, 25°C. Using NMR spectroscopy, the locations of the peptide binding sites were mapped onto the structure of CaM. The binding sites for both peptides were found to overlap with the binding interface for previously identified targets on both domains of CaM. This study demonstrates the plasticity of CaM in target binding and may suggest a possible overlap in target specificity between CaM and the S100 proteins.
钙调蛋白（CaM）是一种多功能信使蛋白，它以钙依赖的方式激活真核细胞中的多种信号通路。有人提出CaM在功能上与S100蛋白不同，S100蛋白是真核细胞钙结合蛋白的一个相关家族。此前，已证明源自肌动蛋白封端蛋白TRTK12和肿瘤抑制蛋白p53的肽与S100蛋白的多个成员相互作用。为了测试这些肽的特异性，使用等温滴定量热法针对人类S100蛋白家族的16个成员以及作为阴性对照的CaM进行筛选。有趣的是，发现TRTK12和p53肽都与CaM相互作用。荧光光谱和核磁共振光谱进一步证实了这些相互作用。这些肽与已知的CaM靶序列具有不同的序列。发现TRTK12肽能独立与CaM的两个结构域相互作用，结合化学计量比为2:1，解离常数Kd,C-term = 2±1μM，Kd,N-term = 14±1μM。相比之下，发现p53肽仅与CaM的C末端结构域相互作用，Kd,C-term = 2±1μM，25°C。使用核磁共振光谱，将肽结合位点的位置映射到CaM的结构上。发现这两种肽的结合位点与CaM两个结构域上先前确定的靶标的结合界面重叠。这项研究证明了CaM在靶标结合方面的可塑性，并可能暗示CaM与S100蛋白在靶标特异性上可能存在重叠。
