Calcium Binding to the Innate Immune Protein Human Calprotectin Revealed by Integrated Mass Spectrometry
Calcium Binding to the Innate Immune Protein Human Calprotectin Revealed by Integrated Mass Spectrometry
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DOI：
10.1021/jacs.9b11950
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发表时间：
2020-08-05
影响因子：
15
通讯作者：
Gross, Michael L.
中科院分区：
文献类型：
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作者：
Adhikari, Jagat;Stephan, Jules R.;Gross, Michael L.
虽然近年来对先天免疫蛋白人钙保护蛋白（hCP）的配位化学和金属保留功能的了解有所扩大，但对其在溶液中的Ca2+结合特性的了解仍然不完整。特别是，Ca2+结合影响结构和增强这种显著的过渡金属隔离蛋白的功能特性的分子基础仍然是谜。为了获得Ca2+结合如何触发hCP寡聚化，增加蛋白酶稳定性和增强抗菌活性的分子图谱，我们实施了一种新的基于集成质谱（MS）的方法，该方法可以很容易地推广到研究其他蛋白质-金属和蛋白质-配体相互作用。三种基于质谱的方法(氢/氘交换质谱动力学；溶液中蛋白质与配体的相互作用，通过质谱、滴定和H/D交换（PLIMSTEX）；和原生质谱)提供了Ca2+结合和寡聚化到hCP的全面分析，而不以任何方式修饰蛋白质。这些方法的整合使我们能够(i)观察作为Ca2+结合位点的hCP的四个区域，（ii）确定结合化学计量为每CP异二聚体4个Ca2+和每CP异四聚体8个Ca2+， （iii）建立导致二聚体到四聚体转变的蛋白质与Ca2+的摩尔比，以及（iv）计算与每个异二聚体的四个Ca2+结合位点相关的结合亲和力。这些定量结果支持hCP以异二聚体形式存在的模型，并且在静止细胞的细胞质中最多与Ca2+半结合。随着释放到细胞外空间，hCP遇到升高的Ca2+浓度并结合更多的Ca2+离子，形成异源四聚体，准备与微生物病原体竞争必需的金属营养素。
Although knowledge of the coordination chemistry and metal-withholding function of the innate immune protein human calprotectin (hCP) has broadened in recent years, understanding of its Ca2+-binding properties in solution remains incomplete. In particular, the molecular basis by which Ca2+ binding affects structure and enhances the functional properties of this remarkable transition-metal-sequestering protein has remained enigmatic. To achieve a molecular picture of how Ca2+ binding triggers hCP oligomerization, increases protease stability, and enhances antimicrobial activity, we implemented a new integrated mass spectrometry (MS)-based approach that can be readily generalized to study other protein-metal and protein-ligand interactions. Three MS-based methods (hydrogen/deuterium exchange MS kinetics; protein-ligand interactions in solution by MS, titration, and H/D exchange (PLIMSTEX); and native MS) provided a comprehensive analysis of Ca2+ binding and oligomerization to hCP without modifying the protein in any way. Integration of these methods allowed us to (i) observe the four regions of hCP that serve as Ca2+-binding sites, (ii) determine the binding stoichiometry to be four Ca2+ per CP heterodimer and eight Ca2+ per CP heterotetramer, (iii) establish the protein-to-Ca2+ molar ratio that causes the dimer-to-tetramer transition, and (iv) calculate the binding affinities associated with the four Ca2+-binding sites per heterodimer. These quantitative results support a model in which hCP exists in its heterodimeric form and is at most halfbound to Ca2+ in the cytoplasm of resting cells. With release into the extracellular space, hCP encounters elevated Ca2+ concentrations and binds more Ca2+ ions, forming a heterotetramer that is poised to compete with microbial pathogens for essential metal nutrients.