ASC Pyrin Domain Self-associates and Binds NLRP3 Protein Using Equivalent Binding Interfaces
ASC Pyrin Domain Self-associates and Binds NLRP3 Protein Using Equivalent Binding Interfaces
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DOI：
10.1074/jbc.m116.741082
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发表时间：
2016-09-09
影响因子：
4.8
通讯作者：
de Alba, Eva
中科院分区：
文献类型：
--
作者：
Oroz, Javier;Barrera-Vilarmau, Susana;de Alba, Eva
死亡结构域超家族成员通常作为接头参与具有重要的细胞凋亡和炎症功能的超分子复合体的组装。这些模块化蛋白由死亡结构域、死亡效应结构域、半胱氨酸酶募集结构域和吡喃结构域组成。尽管它们之间有很高的结构相似性，但只有同型相互作用才能参与复杂的形成，这表明微妙的因素区分了每种相互作用类型。因此，确定这些因素是了解细胞凋亡和炎症的分子基础的关键一步。凋亡相关的SPECK样蛋白含卡蛋白(ASC)和NLRP3通过自身结合和PYD介导的蛋白质-蛋白质相互作用在细胞凋亡和炎症反应中发挥关键作用。为了更好地了解其功能的分子基础，我们用溶液核磁共振光谱和分析超速离心法表征了ASC和NLRP3 PYD的自缔合以及它们的分子间相互作用。我们发现ASC通过等效的蛋白质区域自结合并结合NLRP3 PYD，与后者的结合亲和力更高。这些区域位于蛋白质的两侧，允许形成先前在ASC PYD原纤维组装中所示的多聚体复合体。结果表明，NLRP3 PYD以单体形式存在于溶液中，并以高度有序齐聚的形式存在。尽管如此，我们通过核磁共振确定了其单体的三维溶液结构，并表征了其与ASC PYD的结合。利用我们的新结构数据，我们提出了ASCASC和ASCNLRP3 PYD早期超分子复合体的分子模型，为研究炎症体和细胞凋亡信号的分子机制提供了新的见解。
Death domain superfamily members typically act as adaptors mediating in the assembly of supramolecular complexes with critical apoptosis and inflammation functions. These modular proteins consist of death domains, death effector domains, caspase recruitment domains, and pyrin domains (PYD). Despite the high structural similarity among them, only homotypic interactions participate in complex formation, suggesting that subtle factors differentiate each interaction type. It is thus critical to identify these factors as an essential step toward the understanding of the molecular basis of apoptosis and inflammation. The proteins apoptosis-associated speck-like protein containing a CARD (ASC) and NLRP3 play key roles in the regulation of apoptosis and inflammation through self-association and protein-protein interactions mediated by their PYDs. To better understand the molecular basis of their function, we have characterized ASC and NLRP3 PYD self-association and their intermolecular interaction by solution NMR spectroscopy and analytical ultracentrifugation. We found that ASC self-associates and binds NLRP3 PYD through equivalent protein regions, with higher binding affinity for the latter. These regions are located at opposite sides of the protein allowing multimeric complex formation previously shown in ASC PYD fibril assemblies. We show that NLRP3 PYD coexists in solution as a monomer and highly populated large-order oligomerized species. Despite this, we determined its monomeric three-dimensional solution structure by NMR and characterized its binding to ASC PYD. Using our novel structural data, we propose molecular models of ASCASC and ASCNLRP3 PYD early supramolecular complexes, providing new insights into the molecular mechanisms of inflammasome and apoptosis signaling.