Asymmetric Preorganization of Inverted Pair Residues in the Sodium-Calcium Exchanger.
Asymmetric Preorganization of Inverted Pair Residues in the Sodium-Calcium Exchanger.
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DOI：
10.1038/srep20753
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发表时间：
2016-02-15
影响因子：
4.6
通讯作者：
Khananshvili D
中科院分区：
文献类型：
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作者：
Giladi M;Almagor L;van Dijk L;Hiller R;Man P;Forest E;Khananshvili D
与许多其他蛋白质类似，Na+/Ca~(2+)交换器(NCX)适应重复结构元件的倒置双重对称性，同时通过稳定面向外的构象而表现出功能不对称。在这里，基于结构的突变分析与氢/氢交换质谱仪(HDX-MS)相结合，对简氏甲烷球菌Na+/Ca2+交换因子(NCX_Mj)进行了分析，以确定功能不对称的结构-动力学决定因素。HDX-MS在apo-NCX_Mj的离子配位残基上发现了显著的骨架动力学差异，而Na+或Ca~(2+)与相应位点的结合引起了相对较小但特异的骨架动力学变化。突变分析发现离子配位残基影响催化能力(kcat/Km)，但不影响向外构象的稳定性。相反，不同的“非催化”残基(靠近离子配位残基)控制着外向构象的稳定性，但不能控制催化能力。α1和α2重复序列(在离子结合核心)上的螺旋断裂签名序列(GTSLPE)在折叠/去折叠动力学上有所不同，同时为运输活动提供不对称贡献。目前的数据有力地支持了这样的观点，即无配体离子袋的不对称预组织预先定义了离子结合残基的催化重组，其中与相邻残基的二次相互作用耦合了交替的通道。这些发现为NCX和类似蛋白的离子偶联交替访问提供了结构动力学基础。
In analogy with many other proteins, Na+/Ca2+ exchangers (NCX) adapt an inverted twofold symmetry of repeated structural elements, while exhibiting a functional asymmetry by stabilizing an outward-facing conformation. Here, structure-based mutant analyses of the Methanococcus jannaschii Na+/Ca2+ exchanger (NCX_Mj) were performed in conjunction with HDX-MS (hydrogen/deuterium exchange mass spectrometry) to identify the structure-dynamic determinants of functional asymmetry. HDX-MS identified hallmark differences in backbone dynamics at ion-coordinating residues of apo-NCX_Mj, whereas Na+or Ca2+ binding to the respective sites induced relatively small, but specific, changes in backbone dynamics. Mutant analysis identified ion-coordinating residues affecting the catalytic capacity (kcat/Km), but not the stability of the outward-facing conformation. In contrast, distinct “noncatalytic” residues (adjacent to the ion-coordinating residues) control the stability of the outward-facing conformation, but not the catalytic capacity. The helix-breaking signature sequences (GTSLPE) on the α1 and α2 repeats (at the ion-binding core) differ in their folding/unfolding dynamics, while providing asymmetric contributions to transport activities. The present data strongly support the idea that asymmetric preorganization of the ligand-free ion-pocket predefines catalytic reorganization of ion-bound residues, where secondary interactions with adjacent residues couple the alternating access. These findings provide a structure-dynamic basis for ion-coupled alternating access in NCX and similar proteins.