Multivalent binding of the partially disordered SARS-CoV-2 nucleocapsid phosphoprotein dimer to RNA.
Multivalent binding of the partially disordered SARS-CoV-2 nucleocapsid phosphoprotein dimer to RNA.
复制标题
DOI：
10.1016/j.bpj.2021.03.023
复制
发表时间：
2021-07-20
影响因子：
3.4
通讯作者：
Barbar E
中科院分区：
文献类型：
--
作者：
Forsythe HM;Rodriguez Galvan J;Yu Z;Pinckney S;Reardon P;Cooley RB;Zhu P;Rolland AD;Prell JS;Barbar E
核衣壳蛋白N在SARS冠状病毒感染周期的多个过程中发挥重要作用：保护和包装N组装中的病毒RNA，与刺突蛋白的内部结构域相互作用，在病毒包装和成熟过程中与结构膜(M)蛋白结合，以及与导致感染性病毒颗粒复制的蛋白酶结合。尽管它很重要，但对N蛋白的生物物理研究非常有限，因为它的无序性很高，聚集的倾向很高，而且对自身蛋白分解的敏感性很高。在这里，我们成功地制备了适合生物物理研究的N蛋白和病毒RNA的1000个核苷酸片段。生物物理和生化技术的结合表明，N蛋白是部分无序的，由一个独立折叠的RNA结合域和一个二聚化结构域组成，两侧是无序连接子。该蛋白质组装成紧密的二聚体，二聚化常数为亚微摩尔，但也可以与其他N蛋白形成瞬时相互作用，从而促进更大的低聚物。对RNA100-kDa二聚体蛋白的核磁共振研究发现了一个与1-1000-NTRNA结合的特定结构域，并表明N-∼复合体仍然高度无序。分析超速离心法、等温滴定量热法、多角度光散射和交联实验确定了一种多相混合物，其核心对应于至少70个N二聚体与1-1000个RNA结合。相反，使用类似的实验检测到非常弱的结合，与RNA结合结构域相对应的较小结构。提出了一个解释N的二价结构对其与病毒RNA的多价位点结合的重要性的模型。
The nucleocapsid phosphoprotein N plays critical roles in multiple processes of the severe acute respiratory syndrome coronavirus 2 infection cycle: it protects and packages viral RNA in N assembly, interacts with the inner domain of spike protein, binds to structural membrane (M) protein during virion packaging and maturation, and to proteases causing replication of infective virus particle. Even with its importance, very limited biophysical studies are available on the N protein because of its high level of disorder, high propensity for aggregation, and high susceptibility for autoproteolysis. Here, we successfully prepare the N protein and a 1000-nucleotide fragment of viral RNA in large quantities and purity suitable for biophysical studies. A combination of biophysical and biochemical techniques demonstrates that the N protein is partially disordered and consists of an independently folded RNA-binding domain and a dimerization domain, flanked by disordered linkers. The protein assembles as a tight dimer with a dimerization constant of sub-micromolar but can also form transient interactions with other N proteins, facilitating larger oligomers. NMR studies on the ∼100-kDa dimeric protein identify a specific domain that binds 1–1000-nt RNA and show that the N-RNA complex remains highly disordered. Analytical ultracentrifugation, isothermal titration calorimetry, multiangle light scattering, and cross-linking experiments identify a heterogeneous mixture of complexes with a core corresponding to at least 70 dimers of N bound to 1–1000 RNA. In contrast, very weak binding is detected with a smaller construct corresponding to the RNA-binding domain using similar experiments. A model that explains the importance of the bivalent structure of N to its binding on multivalent sites of the viral RNA is presented.
登录
查看更多内容
影响因子：
4.1
作者：
Chukwudozie OS;Chukwuanukwu RC;Iroanya OO;Eze DM;Duru VC;Dele-Alimi TO;Kehinde BD;Bankole TT;Obi PC;Okinedo EU
通讯作者：
Okinedo EU
影响因子：
2.9
作者：
Huang, QL;Yu, LP;Olejniczak, ET
通讯作者：
Olejniczak, ET
影响因子：
4.1
作者：
Frey, Steffen;Goerlich, Dirk
通讯作者：
Goerlich, Dirk
影响因子：
7.6
作者：
Chang CK;Hou MH;Chang CF;Hsiao CD;Huang TH
通讯作者：
Huang TH
影响因子：
4.6
作者：
Cong Y;Kriegenburg F;de Haan CAM;Reggiori F
通讯作者：
Reggiori F