Abstract:
The nucleocapsid protein (N) of SARS-CoV-2 is essential for virus replication, genome packaging, evading host immunity, and virus maturation. N is a multidomain protein composed of an independently folded monomeric N-terminal domain that is the primary site for RNA binding and a dimeric C-terminal domain that is essential for efficient phase separation and condensate formation with RNA. The domains are separated by a disordered Ser/Arg-rich region preceding a self-associating Leu-rich helix. Phosphorylation in the Ser/Arg region in infected cells decreases the viscosity of N:RNA condensates promoting viral replication and host immune evasion. The molecular level effect of phosphorylation, however, is missing from our current understanding. Using NMR spectroscopy and analytical ultracentrifugation, we show that phosphorylation destabilizes the self-associating Leu-rich helix 30 amino-acids distant from the phosphorylation site. NMR and gel shift assays demonstrate that RNA binding by the linker is dampened by phosphorylation, whereas RNA binding to the full-length protein is not significantly affected presumably due to retained strong interactions with the primary RNA-binding domain. Introducing a switchable self-associating domain to replace the Leu-rich helix confirms the importance of linker self-association to droplet formation and suggests that phosphorylation not only increases solubility of the positively charged elongated Ser/Arg region as observed in other RNA-binding proteins but can also inhibit self-association of the Leu-rich helix. These data highlight the effect of phosphorylation both at local sites and at a distant self-associating hydrophobic helix in regulating liquid-liquid phase separation of the entire protein.
摘要:
SARS-CoV-2的核衣壳蛋白(N)对于病毒复制至关重要,基因组包装,逃避宿主免疫,和病毒成熟。N是由独立折叠的单体N末端结构域组成的多域蛋白,是RNA结合的主要位点。和二聚体C末端结构域,这对于有效的相分离和与RNA的缩合物形成是必不可少的。结构域被自缔合的富Leu螺旋之前的无序的富Ser/Arg区域分开。感染细胞中Ser/Arg区域的磷酸化降低了促进病毒复制和宿主免疫逃避的N:RNA缩合物的粘度。磷酸化的分子水平效应,然而,我们目前的理解中缺失了。使用NMR光谱和分析超速离心,我们表明磷酸化使远离磷酸化位点的自缔合富含Leu的螺旋30个氨基酸不稳定。NMR和凝胶移位测定表明,通过接头结合的RNA被磷酸化抑制,而RNA与全长蛋白的结合没有显著影响,推测是由于与初级RNA结合结构域保留了强相互作用。引入可切换的自缔合结构域来替换富Leu螺旋证实了接头自缔合对液滴形成的重要性,并且表明磷酸化不仅增加了如在其他RNA结合蛋白中观察到的带正电荷的延长的Ser/Arg区的溶解度,而且还可以抑制富Leu螺旋的自缔合。这些数据强调了局部位点和远处自缔合疏水螺旋的磷酸化在调节整个蛋白质的液-液相分离中的作用。