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首页> 外文期刊>Journal of Molecular Biology >Solution structure and characterisation of the human pyruvate dehydrogenase complex core assembly.
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Solution structure and characterisation of the human pyruvate dehydrogenase complex core assembly.

机译：人丙酮酸脱氢酶复合物核心组件的溶液结构和表征。

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Mammalian pyruvate dehydrogenase complex (PDC) is a key multi-enzyme assembly that is responsible for glucose homeostasis maintenance and conversion of pyruvate into acetyl-CoA. It comprises a central pentagonal dodecahedral core consisting of two subunit types (E2 and E3BP) to which peripheral enzymes (E1 and E3) bind tightly but non-covalently. Currently, there are two conflicting models of PDC (E2+E3BP) core organisation: the 'addition' model (60+12) and the 'substitution' model (48+12). Here we present the first ever low-resolution structures of human recombinant full-length PDC core (rE2/E3BP), truncated PDC core (tE2/E3BP) and native bovine heart PDC core (bE2/E3BP) obtained by small-angle X-ray scattering and small-angle neutron scattering. These structures, corroborated by negative-stain and cryo electron microscopy data, clearly reveal open pentagonal core faces, favouring the 'substitution' model of core organisation. The native and recombinant core structures are all similar to the truncated bacterial E2 core crystal structure obtained previously. Cryo-electron microscopy reconstructions of rE2/E3BP and rE2/E3BP:E3 directly confirm that the core has open pentagonal faces, agree with scattering-derived models and show density extending outwards from their surfaces, which is much more structurally ordered in the presence of E3. Additionally, analytical ultracentrifugation characterisation of rE2/E3BP, rE2 (full-length recombinant E2-only) and tE2/E3BP supports the substitution model. Superimposition of the small-angle neutron scattering tE2/E3BP and truncated bacterial E2 crystal structures demonstrates conservation of the overall pentagonal dodecahedral morphology, despite evolutionary diversity. In addition, unfolding studies using circular dichroism and tryptophan fluorescence spectroscopy show that the rE2/E3BP is less stable than its rE2 counterpart, indicative of a role for E3BP in core destabilisation. The architectural complexity and lower stability of the E2/E3BP core may be of benefit to mammals, where sophisticated fine-tuning is required for cores with optimal catalytic and regulatory efficiencies.
机译：哺乳动物丙酮酸脱氢酶复合物（PDC）是关键的多酶组装体，负责葡萄糖的体内稳态维持以及将丙酮酸转化为乙酰CoA。它包含由两个亚基类型（E2和E3BP）组成的中央五边形十二面体核心，外围酶（E1和E3）紧密但非共价结合。当前，PDC（E2 + E3BP）核心组织存在两种相互冲突的模型：“加法”模型（60 + 12）和“替代”模型（48 + 12）。在这里，我们介绍了人类重组全长PDC核心（rE2 / E3BP），截短的PDC核心（tE2 / E3BP）和天然牛心PDC核心（bE2 / E3BP）的第一个低分辨率结构，该结构通过小角度X-射线散射和小角度中子散射。这些结构由负染色和低温电子显微镜数据证实，清楚地揭示了开放的五边形核心面，有利于核心组织的“替代”模型。天然和重组核心结构均类似于先前获得的截短的细菌E2核心晶体结构。 rE2 / E3BP和rE2 / E3BP：E3的低温电子显微镜重建直接证实了核具有开放的五边形面，与散射衍生的模型一致，并且显示了从其表面向外延伸的密度，这在存在下的情况下在结构上更加有序E3。另外，rE2 / E3BP，rE2（仅全长重组E2）和tE2 / E3BP的分析超速离心特性支持替代模型。小角中子散射tE2 / E3BP和截短的细菌E2晶体结构的叠加表明，尽管进化多样性，但总体上保留了五边形十二面体形态。此外，使用圆二色性和色氨酸荧光光谱法进行的展开研究表明，rE2 / E3BP的稳定性低于其rE2对应物，表明E3BP在核心不稳定中的作用。 E2 / E3BP核的结构复杂性和较低的稳定性可能对哺乳动物有利，因为对于具有最佳催化和调节效率的核，需要进行复杂的微调。

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