Protein Biophysics, Institute for Molecules and Materials, Radboud University Nijmegen, Nijmegen, The Netherlands.
Proteins. 2010 Jun;78(8):1813-24. doi: 10.1002/prot.22695.
We report the effects of Ca(2+) binding on the backbone relaxation rates and chemical shifts of the AD and BD splice variants of the second Ca(2+)-binding domain (CBD2) of the sodium-calcium exchanger. Analysis of the Ca(2+)-induced chemical shifts perturbations yields similar K(D) values of 16-24 microM for the two CBD2-AD Ca(2+)-binding sites, and significant effects are observed up to 20 A away. To quantify the Ca(2+)-induced chemical shift changes, we performed a comparative analysis of eight Ca(2+)-binding proteins that revealed large differences between different protein folds. The CBD2 (15)N relaxation data show the CBD2-AD Ca(2+) coordinating loops to be more rigid in the Ca(2+)-bound state as well as to affect the FG-loop located at the opposite site of the domain. The equivalent loops of the CBD2-BD splice variant do not bind Ca(2+) and are much more dynamic relative to both the Ca(2+)-bound and apo forms of CBD2-AD. A more structured FG-loop in CBD2-BD is suggested by increased S(2) order parameter values relative to both forms of CBD2-AD. The chemical shift and relaxation data together indicate that, in spite of the small structural changes, the Ca(2+)-binding event is felt throughout the molecule. The data suggest that the FG-loop plays an important role in connecting the Ca(2+)-binding event with the other cytosolic domains of the NCX, in line with in vivo and in vitro biochemical data as well as modeling results that connect the CBD2 FG-loop with the first Ca(2+)-binding domain of NCX.
我们报告了钙离子结合对钠钙交换体第二钙结合域（CBD2）的 AD 和 BD 剪接变体的骨架弛豫率和化学位移的影响。对 Ca(2+)诱导的化学位移扰动的分析为两个 CBD2-AD Ca(2+)结合位点产生了相似的 K(D)值为 16-24 microM，并且在 20 A 以外的距离观察到了显著的影响。为了量化 Ca(2+)诱导的化学位移变化，我们对 8 种 Ca(2+)结合蛋白进行了比较分析，结果表明不同蛋白折叠之间存在很大差异。CBD2 (15)N 弛豫数据表明，在 Ca(2+)结合状态下，CBD2-AD Ca(2+)配位环更加刚性，并影响位于域对面的 FG-环。CBD2-BD 剪接变体的等效环不结合 Ca(2+)，并且相对于 CBD2-AD 的 Ca(2+)结合和无钙形式，其动态性要大得多。CBD2-BD 中 FG-环的 S(2)序参数值相对于 CBD2-AD 的两种形式都有所增加，表明 FG-环的结构更加有序。化学位移和弛豫数据表明，尽管结构变化很小，但 Ca(2+)结合事件贯穿整个分子。这些数据表明，FG-环在将 Ca(2+)结合事件与 NCX 的其他胞质域连接方面起着重要作用，这与体内和体外生化数据以及将 CBD2 FG-环与 NCX 的第一个 Ca(2+)结合域连接起来的建模结果一致。
