Dissecting Interactions between RNA and Coronavirus Nucleocapsid Proteins Using Native Mass Spectrometry

IF 2.7 2区 化学 Q2 BIOCHEMICAL RESEARCH METHODS
Virginia K. James, Olivia E. Dioli and Jennifer S. Brodbelt*, 
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Abstract

The nucleocapsid protein is the most abundant protein in the SARS-CoV virus and plays critical roles in RNA packaging and storage, yet the nature of the protein’s interactions with RNA remain elusive owing to the presence of multiple disordered regions of the protein. Here we evaluate the protein–RNA and protein–protein interactions of nucleocapsid proteins from the SARS-CoV-1 WT, SARS-CoV-2 WT, and SARS-CoV-1 omicron to better understand how the interactions are modulated by sequence variations. By using native mass spectrometry, we reveal that all three nucleocapsid proteins exist as both monomers and dimers that bind up to two RNA molecules per monomer, suggesting dual RNA binding sites. Ultraviolet photodissociation (UVPD) was used to localize the RNA binding sites found in the RNA binding domain and N-terminus of the nucleocapsid protein. The stabilities of the dimer complexes of the three nucleocapsid were monitored using energy-variable collision-induced dissociation, revealing that the dimer from SARS-CoV-1 is less stable than the dimers of the other two SARS-CoV variants in the gas phase. Furthermore, variable temperature electrospray ionization (ESI) experiments of the RNA-bound nucleocapsid proteins indicated that the protein–RNA interactions are stronger than the protein–protein interactions in solution as the dimeric protein–RNA complexes dissociate into monomeric protein–RNA complexes prior to the loss of RNA.

Abstract Image

利用天然质谱分析RNA与冠状病毒核衣壳蛋白之间的相互作用。
核衣壳蛋白是SARS-CoV病毒中最丰富的蛋白质,在RNA的包装和储存中起着关键作用,但由于该蛋白存在多个无序区域,因此该蛋白与RNA相互作用的性质仍然难以捉摸。在这里,我们评估了来自SARS-CoV-1 WT、SARS-CoV-2 WT和SARS-CoV-1组粒的核衣壳蛋白的蛋白质- rna和蛋白质-蛋白质相互作用,以更好地了解这些相互作用是如何被序列变化调节的。通过使用天然质谱法,我们发现所有三种核衣壳蛋白都以单体和二聚体的形式存在,每个单体可以结合两个RNA分子,表明存在双RNA结合位点。紫外光解作用(UVPD)用于定位核衣壳蛋白的RNA结合域和n端RNA结合位点。利用能量可变碰撞诱导解离法监测了三种核衣壳二聚体复合物的稳定性,揭示了SARS-CoV-1二聚体在气相中的稳定性低于其他两种SARS-CoV变体的二聚体。此外,RNA结合的核衣壳蛋白的变温电喷雾电离(ESI)实验表明,由于二聚体蛋白质-RNA复合物在RNA丢失之前解离成单体蛋白质-RNA复合物,因此蛋白质-RNA相互作用比蛋白质-蛋白质相互作用在溶液中更强。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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来源期刊
CiteScore
5.50
自引率
9.40%
发文量
257
审稿时长
1 months
期刊介绍: The Journal of the American Society for Mass Spectrometry presents research papers covering all aspects of mass spectrometry, incorporating coverage of fields of scientific inquiry in which mass spectrometry can play a role. Comprehensive in scope, the journal publishes papers on both fundamentals and applications of mass spectrometry. Fundamental subjects include instrumentation principles, design, and demonstration, structures and chemical properties of gas-phase ions, studies of thermodynamic properties, ion spectroscopy, chemical kinetics, mechanisms of ionization, theories of ion fragmentation, cluster ions, and potential energy surfaces. In addition to full papers, the journal offers Communications, Application Notes, and Accounts and Perspectives
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