Coronavirus (CoV) nonstructural proteins (nsps) assemble to form the replication-transcription complex (RTC) responsible for viral RNA synthesis. nsp7 and nsp8 are important cofactors of the RTC, as they interact and regulate the activity of RNA-dependent RNA polymerase and other nsps. To date, no structure of the full-length SARS-CoV-2 nsp7:nsp8 complex has been published. The current understanding of this complex is based on structures from truncated constructs, with missing electron densities, or from related CoV species where SARS-CoV-2 nsp7 and nsp8 share upward of 90% sequence identity. Despite available structures solved using crystallography and cryo-EM representing detailed static snapshots of the nsp7:nsp8 complex, it is evident that the complex has a high degree of structural plasticity. However, relatively little is known about the conformational dynamics of the individual proteins and how they complex to interact with other nsps. Here, the solution-based structural proteomic techniques, hydrogen-deuterium exchange mass spectrometry (HDX-MS) and cross-linking mass spectrometry (XL-MS), illuminate the dynamics of SARS-CoV-2 full-length nsp7 and nsp8 proteins and the nsp7:nsp8 protein complex. Results presented from the two techniques are complementary and validate the interaction surfaces identified from the published three-dimensional heterotetrameric crystal structure of the SARS-CoV-2 truncated nsp7:nsp8 complex. Furthermore, mapping of XL-MS data onto higher-order complexes suggests that SARS-CoV-2 nsp7 and nsp8 do not assemble into a hexadecameric structure as implied by the SARS-CoV full-length nsp7:nsp8 crystal structure. Instead, our results suggest that the nsp7:nsp8 heterotetramer can dissociate into a stable dimeric unit that might bind to nsp12 in the RTC without significantly altering nsp7-nsp8 interactions.

中文翻译：

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使用结构蛋白质组学揭示 SARS-CoV-2 nsp7 和 nsp8 的结构可塑性。

冠状病毒 (CoV) 非结构蛋白 (nsps) 组装形成负责病毒 RNA 合成的复制转录复合物 (RTC)。nsp7 和 nsp8 是 RTC 的重要辅助因子，因为它们相互作用并调节 RNA 依赖性 RNA 聚合酶和其他 nsp 的活性。迄今为止，尚未公布全长 SARS-CoV-2 nsp7:nsp8 复合物的结构。目前对该复合物的理解是基于缺失电子密度的截短构建体的结构，或来自相关 CoV 物种的结构，其中 SARS-CoV-2 nsp7 和 nsp8 具有 90% 以上的序列同一性。尽管使用晶体学和冷冻电镜解析了可用的结构，代表了 nsp7:nsp8 复合物的详细静态快照，但显然该复合物具有高度的结构可塑性。然而，对于单个蛋白质的构象动力学以及它们如何与其他 nsps 相互作用形成复合物，人们知之甚少。在此，基于解决方案的结构蛋白质组学技术、氢-氘交换质谱 (HDX-MS) 和交联质谱 (XL-MS) 阐明了 SARS-CoV-2 全长 nsp7 和 nsp8 蛋白的动态变化，以及nsp7:nsp8 蛋白质复合物。两种技术得出的结果是互补的，并验证了从已发表的 SARS-CoV-2 截短的 nsp7:nsp8 复合物的三维异四聚体晶体结构中鉴定出的相互作用表面。此外，将 XL-MS 数据映射到高阶复合物上表明，SARS-CoV-2 nsp7 和 nsp8 不会组装成十六聚体结构，如 SARS-CoV 全长 nsp7:nsp8 晶体结构所暗示的那样。相反，我们的结果表明，nsp7:nsp8 异四聚体可以解离成稳定的二聚体单元，该二聚体单元可能与 RTC 中的 nsp12 结合，而不会显着改变 nsp7-nsp8 相互作用。