The COVID‐2019 pandemic is the most severe acute public health threat of the twenty‐first century. To properly address this crisis with both robust testing and novel treatments, we require a deep understanding of the life cycle of the causative agent, the SARS‐CoV‐2 coronavirus. Here, we examine the architecture and self‐assembly properties of the SARS‐CoV‐2 nucleocapsid protein, which packages viral RNA into new virions. We determined a 1.4 Å resolution crystal structure of this protein's N2b domain, revealing a compact, intertwined dimer similar to that of related coronaviruses including SARS‐CoV. While the N2b domain forms a dimer in solution, addition of the C‐terminal spacer B/N3 domain mediates formation of a homotetramer. Using hydrogen‐deuterium exchange mass spectrometry, we find evidence that at least part of this putatively disordered domain is structured, potentially forming an α‐helix that self‐associates and cooperates with the N2b domain to mediate tetramer formation. Finally, we map the locations of amino acid substitutions in the N protein from over 38,000 SARS‐CoV‐2 genome sequences. We find that these substitutions are strongly clustered in the protein's N2a linker domain, and that substitutions within the N1b and N2b domains cluster away from their functional RNA binding and dimerization interfaces. Overall, this work reveals the architecture and self‐assembly properties of a key protein in the SARS‐CoV‐2 life cycle, with implications for both drug design and antibody‐based testing.

中文翻译：

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SARS-CoV-2 核衣壳蛋白的结构和自组装。

2019 年新冠肺炎大流行是二十一世纪最严重的公共卫生威胁。为了通过强有力的检测和新颖的治疗方法正确应对这场危机，我们需要深入了解病原体 SARS-CoV-2 冠状病毒的生命周期。在这里，我们研究了 SARS-CoV-2 核衣壳蛋白的结构和自组装特性，该蛋白将病毒 RNA 包装成新的病毒体。我们确定了该蛋白 N2b 结构域的 1.4 Å 分辨率晶体结构，揭示了一种紧凑的、相互缠绕的二聚体，类似于包括 SARS-CoV 在内的相关冠状病毒。虽然 N2b 结构域在溶液中形成二聚体，但添加 C 末端间隔区 B/N3 结构域会介导同源四聚体的形成。使用氢-氘交换质谱，我们发现证据表明，这个假定的无序结构域的至少一部分是结构化的，可能形成一个 α 螺旋，该螺旋与 N2b 结构域自缔合并协同介导四聚体形成。最后，我们从 38,000 多个 SARS-CoV-2 基因组序列中绘制了 N 蛋白中氨基酸取代的位置。我们发现这些取代强烈聚集在蛋白质的 N2a 接头结构域中，而 N1b 和 N2b 结构域内的取代则远离其功能性 RNA 结合和二聚化界面。总的来说，这项工作揭示了 SARS-CoV-2 生命周期中关键蛋白质的结构和自组装特性，对药物设计和基于抗体的测试都有影响。