Cu(II) EPR Reveals Two Distinct Binding Sites and Oligomerization of Innate Immune Protein Calgranulin C
Cu(II) EPR Reveals Two Distinct Binding Sites and Oligomerization of Innate Immune Protein Calgranulin C
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DOI：
10.1007/s00723-018-1053-7
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发表时间：
2018-11-01
影响因子：
1
通讯作者：
Saxena, Sunil
中科院分区：
文献类型：
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作者：
Ghosh, Shreya;Garcia, Velia;Saxena, Sunil
S100 A12或钙粒蛋白C是EF-手型钙调节蛋白的S100家族的同源二聚体抗微生物蛋白。S100 A12参与许多疾病，如炎症、肿瘤侵袭、癌症和神经系统疾病如阿尔茨海默病。过渡金属离子与蛋白质的结合是重要的，因为金属离子的螯合诱导蛋白质的构象变化，抑制各种病原微生物的生长。在这项工作中，我们探测钙颗粒蛋白C的Cu 2+结合特性。我们证明了钙粒蛋白C中的两个Cu 2+结合位点在溶液中显示出不同的配位环境。连续波电子自旋共振（CW-ESR）光谱的Cu 2+结合蛋白质清楚地显示两个不同的组分在较高的Cu 2+：蛋白质的比例，这是两个不同的结合环境的Cu 2+离子的指示。G（||），A（||）值对于两种组分也是不同的，表明在每个位点中直接配位的氮的数目不同。此外，我们进行CW-ESR滴定，以获得的Ca 2+负载的蛋白质的结合亲和力的Cu 2+离子。我们观察到一个积极的协同性结合的两个Cu 2+离子。为了进一步探测Cu 2+配位，我们还进行了电子自旋回波包络调制（ESEEM）实验。我们在两个不同的领域进行ESEEM，其中一个Cu 2+结合位点占主导地位的其他。在这两个网站，我们看到不同的签名Cu 2 +-组氨酸的协调。然而，我们清楚地看到，对应于两个Cu 2+结合位点的ESEEM光谱是显著不同的。在两个不同的场作用下，双量子峰的强度相对于核四极相互作用峰有明显的变化。此外，ESEEM沿着与超精细亚能级相关表明，只有一个的两个Cu 2+结合位点的骨干配位，证实了我们以前的观察。最后，我们进行双电子-电子共振光谱探测结合环境的差异是否是由于Cu 2+结合到蛋白质中的不同位点。我们得到了一个距离分布，在3 nm处有一个尖锐的峰，在4 nm处有一个宽峰。较短的距离与预期的二聚体从晶体结构的Cu 2 +-Cu 2+距离一致。较长的距离与发生低聚时的Cu 2 +-Cu 2+距离一致。
S100A12 or Calgranulin C is a homodimeric antimicrobial protein of the S100 family of EF-hand calcium-modulated proteins. S100A12 is involved in many diseases such as inflammation, tumor invasion, cancer and neurological disorders such as Alzheimer's disease. The binding of transition metal ions to the protein is important as the sequestering of the metal ion induces conformational changes in the protein, inhibiting the growth of various pathogenic microorganisms. In this work, we probe the Cu2+ binding properties of Calgranulin C. We demonstrate that the two Cu2+ binding sites in Calgranulin C show different coordination environments in solution. Continuous wave-electron spin resonance(CW-ESR) spectra of Cu2+-bound protein clearly show two distinct components at higher Cu2+:protein ratios, which is indicative of the two different binding environments for the Cu2+ ions. The g(||) and A(||) values are also different for the two components, indicating that the number of directly coordinated nitrogen in each site differs. Furthermore, we perform CW-ESR titrations to obtain the binding affinity of the Ca2+-loaded protein to Cu2+ ions. We observe a positive cooperativity in binding of the two Cu2+ ions. To further probe the Cu2+ coordination, we also perform electron spin echo envelope modulation (ESEEM) experiment. We perform ESEEM at two different fields where one Cu2+ binding site dominates the other. At both sites we see distinct signatures of Cu2+-histidine coordination. However, we clearly see that the ESEEM spectra corresponding to the two Cu2+ binding sites are significantly different. There is clear change in the intensity of the double quantum peak with respect to the nuclear quadrupole interaction peak at the two different fields. Furthermore, ESEEM along with hyperfine sublevel correlation show that only one of the two Cu2+ binding sites has backbone coordination, confirming our previous observation. Finally, we perform double electron-electron resonance spectroscopy to probe if the difference in binding environment is due to the Cu2+ binding to different sites in the protein. We obtain a distance distribution with a sharp peak at 3nm and a broad peak at 4nm. The shorter distance agrees with the Cu2+-Cu2+ distance expected for a dimer from the crystal structure. The longer distance is consistent with the Cu2+-Cu2+ distance when oligomerization occurs.