Creon Anne, Josts Inokentijs, Niebling Stephan, Huse Nils, Tidow Henning
Struct Dyn. 2018 Nov 7;5(6):064701. doi: 10.1063/1.5053466. eCollection 2018 Nov.
Calmodulin (CaM) is a very conserved, ubiquitous, eukaryotic protein that binds four Ca ions with high affinity. It acts as a calcium sensor by translating Ca signals into cellular processes such as metabolism, inflammation, immune response, memory, and muscle contraction. Calcium binding to CaM leads to conformational changes that enable Ca/CaM to recognize and bind various target proteins with high affinity. The binding mode and binding partners of CaM are very diverse, and a consensus binding sequence is lacking. Here, we describe an elegant system that allows conformation-specific detection of CaM-binding to its binding partners. We incorporate the unnatural amino acid p-azido-phenylalanine (AzF) in different positions of CaM and follow its unique spectral signature by infrared (IR)-spectroscopy of the azido stretching vibration. Our results suggest that the AzF vibrational probe is sensitive to the chemical environment in different CaM/CaM-binding domain (CaMBD) complexes, which allows differentiating between different binding motifs according to the spectral characteristics of the azido stretching mode. We corroborate our results with a crystal structure of AzF-labelled CaM (CaM108AzF) in complex with a binding peptide from calmodulin-dependent protein kinase IIα identifying the structural basis for the observed IR frequency shifts.
钙调蛋白（CaM）是一种高度保守、广泛存在的真核蛋白，能以高亲和力结合4个钙离子。它作为一种钙传感器，通过将钙信号转化为细胞过程，如新陈代谢、炎症、免疫反应、记忆和肌肉收缩。钙与CaM结合会导致构象变化，使Ca/CaM能够以高亲和力识别并结合各种靶蛋白。CaM的结合模式和结合伙伴非常多样，且缺乏一致的结合序列。在此，我们描述了一个精巧的系统，该系统能够对CaM与其结合伙伴的结合进行构象特异性检测。我们将非天然氨基酸对叠氮基苯丙氨酸（AzF）掺入CaM的不同位置，并通过叠氮基伸缩振动的红外（IR）光谱追踪其独特的光谱特征。我们的结果表明，AzF振动探针对不同CaM/钙调蛋白结合域（CaMBD）复合物中的化学环境敏感，这使得能够根据叠氮基伸缩模式的光谱特征区分不同的结合基序。我们通过AzF标记的CaM（CaM108AzF）与钙调蛋白依赖性蛋白激酶IIα的结合肽形成复合物的晶体结构证实了我们的结果，确定了观察到的红外频率偏移的结构基础。
