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Abstract：
Structure and functions of S100 proteins are regulated by two distinct calcium binding EF hand motifs. In this work, we used solution-state NMR spectroscopy to investigate the cooperativity between the two calcium binding sites and map the allosteric changes at the target binding site. To parse the contribution of the individual calcium binding events, variants of S100A12 were designed to selectively bind calcium to either the EF-I (N63A) or EF-II (E31A) loop, respectively. Detailed analysis of the backbone chemical shifts for wildtype protein and its mutants indicates that calcium binding to the canonical EF-II loop is the principal trigger for the conformational switch between \'closed\' apo to the \'open\' Ca2+ -bound conformation of the protein. Elimination of binding in S100-specific EF-I loop has limited impact on the calcium binding affinity of the EF-II loop and the concomitant structural rearrangement. In contrast, deletion of binding in the EF-II loop significantly attenuates calcium affinity in the EF-I loop and the structure adopts a \'closed\' apo-like conformation. Analysis of experimental amide nitrogen (15 N) relaxation rates (R1 , R2 , and 15 N-{1 H} NOE) and molecular dynamics (MD) simulations demonstrate that the calcium bound state is relatively floppy with pico-nanosecond motions induced in functionally relevant domains responsible for target recognition such as the hinge domain and the C-terminal residues. Experimental relaxation studies combined with MD simulations show that while calcium binding in the EF-I loop alone does not induce significant motions in the polypeptide chain, EF-I regulates fluctuations in the polypeptide in the presence of bound calcium in the EF-II loop. These results offer novel insights into the dynamic regulation of target recognition by calcium binding and unravels the role of cooperativity between the two calcium binding events in S100A12.
摘要：
S100蛋白的结构和功能由两个不同的钙结合EF手基序调节。在这项工作中,我们使用溶液状态NMR光谱研究了两个钙结合位点之间的协同性,并绘制了靶结合位点的变构变化图.为了解析各个钙结合事件的贡献，S100A12的变体被设计为选择性地将钙与EF-I（N63A）或EF-II（E31A）环结合，分别。对野生型蛋白及其突变体的主链化学位移的详细分析表明，钙与规范的EF-II环的结合是“封闭的”apo与“开放的”Ca2结合的构象之间构象转换的主要触发因素。蛋白质的构象。消除S100特异性EF-I环中的结合对EF-II环的钙结合亲和力和伴随的结构重排具有有限的影响。相比之下,EF-II环中结合的缺失显着减弱了EF-I环中的钙亲和力，并且该结构采用“闭合”apo样构象。实验酰胺氮（15N）弛豫率的分析（R1，R2,和15N-{1H}NOE）和分子动力学（MD）模拟表明，钙结合态相对松弛，在负责靶标识别的功能相关域（例如铰链域和C-末端残基）中诱导了皮纳秒运动。与MD模拟相结合的实验松弛研究表明，尽管EF-I环中的钙结合单独不会诱导多肽链中的显着运动，EF-I在EF-II环中结合钙的存在下调节多肽的波动。这些结果为钙结合对靶标识别的动态调节提供了新的见解，并揭示了S100A12中两个钙结合事件之间的协同作用。
