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首页> 外文期刊>Journal of Molecular Biology >Flexible Stoichiometry and Asymmetry of the PIDDosome Core Complex by Heteronuclear NMR Spectroscopy and Mass Spectrometry
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Flexible Stoichiometry and Asymmetry of the PIDDosome Core Complex by Heteronuclear NMR Spectroscopy and Mass Spectrometry

机译：核磁共振波谱和质谱法对PIDDosome核配合物的柔性化学计量和不对称性

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摘要

Homotypic death domain (DD)-DD interactions are important in the assembly of oligomeric signaling complexes such as the PIDDosome that acts as a platform for activation of caspase-2-dependent apoptotic signaling. The structure of the PIDDosome core complex exhibits an asymmetric three-layered arrangement containing five PIDD-DDs in one layer, five RAIDD-DDs in a second layer and an additional two RAIDD-DDs. We addressed complex formation between PIDD-DD and RAIDD-DD in solution using heteronuclear nuclear magnetic resonance (NMR) spectroscopy, nanoflow electrospray ionization mass spectrometry and size-exclusion chromatography with multi-angle light scattering. The DDs assemble into complexes displaying molecular masses in the range 130-158 kDa and RAIDD-DD: PIDD-DD stoichiometries of 5:5, 6:5 and 7:5. These data suggest that the crystal structure is representative of only the heaviest species in solution and that two RAIDD-DDs are loosely attached to the 5:5 core. Two-dimensional H-1,N-15-NMR experiments exhibited signal loss upon complexation consistent with the formation of high-molecular-weight species. C-13-Methyl-transverse relaxation optimized spectroscopy measurements of the PIDDosome core exhibit signs of differential line broadening, cross-peak splitting and chemical shift heterogeneity that reflect the presence of non-equivalent sites at interfaces within an asymmetric complex. Experiments using a mutant RAIDD-DD that forms a monodisperse 5:5 complex with PIDD-DD show that the spectroscopic signature derives from the quasi- but non-exact equivalent environments of each DD. Since this characteristic was previously demonstrated for the complex between the DDs of CD95 and FADD, the NMR data for this system are consistent with the formation of a structure homologous to the PIDDosome core. (C) 2014 The Authors. Published by Elsevier Ltd.
机译：同型死亡域（DD）-DD相互作用在寡聚信号复合物（如PIDDosome）的组装中很重要，该复合物充当激活caspase-2依赖性细胞凋亡信号传导的平台。 PIDDosome核心复合体的结构呈现出不对称的三层结构，其中一层包含五个PIDD-DD，第二层包含五个RAIDD-DD，另外两个RAIDD-DD。我们使用异核核磁共振（NMR）光谱，纳流电喷雾电离质谱和具有多角度光散射的尺寸排阻色谱法解决了溶液中PIDD-DD和RAIDD-DD之间的复杂形成。 DD组装成显示130-158 kDa范围内的分子质量的复合物，RAIDD-DD：PIDD-DD化学计量比为5：5、6：5和7：5。这些数据表明晶体结构仅代表溶液中最重的物质，并且两个RAIDD-DD松散地附着在5：5核上。二维H-1，N-15-NMR实验表明，络合后的信号损失与高分子量物质的形成一致。 PIDDosome核心的C-13-甲基横向弛豫优化光谱测量结果显示出差分谱线展宽，交叉峰分裂和化学位移异质性的迹象，反映出不对称复合物中界面处存在非等价位。使用突变的RAIDD-DD与PIDD-DD形成5：5单分散复合物的实验表明，光谱特征来自每个DD的准但非精确等效环境。由于先前已针对CD95和FADD的DD之间的复合物证明了该特性，因此该系统的NMR数据与形成与PIDDosome核同源的结构一致。 （C）2014作者。由Elsevier Ltd.发布

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