Mass spectrometric based detection of protein nucleotidylation in the RNA polymerase of SARS-CoV-2
Mass spectrometric based detection of protein nucleotidylation in the RNA polymerase of SARS-CoV-2
复制标题
基于质谱的 SARS-CoV-2 RNA 聚合酶中蛋白质核苷酸化检测
DOI：
10.1101/2020.10.07.330324
复制
发表时间：
2021
影响因子：
5.9
通讯作者：
Sussman, Michael R.
中科院分区：
文献类型：
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作者：
Conti, Brian J;Leicht, Andrew S;Kirchdoerfer, Robert N;Sussman, Michael R.
冠状病毒，如严重急性呼吸综合征冠状病毒2型（SARS-CoV-2），在RNA依赖的RNA聚合酶内的结构蛋白（nsp）12蛋白的N-末端（NiRAN）结构域编码核苷酸转移酶。在这里，我们显示了检测鸟苷一磷酸（GMP）和尿苷一磷酸修饰的氨基酸在巢状病毒蛋白使用重同位素辅助质谱（MS）和MS/MS肽测序。我们发现马动脉炎病毒（EAV）蛋白nsp 7中的赖氨酸-143是通过磷酰胺键体外GMP附着的主要位点。在SARS-CoV-2复制酶蛋白中，我们证明了nsp 12介导的nsp 7赖氨酸-2的核苷酸化。我们的研究结果表明，新的策略检测GMP-肽连接，可以适用于更高的通量筛选使用质谱技术。预计这些数据对于快速和及时表征SARS-CoV-2中的新酶活性是重要的，该酶活性可能是旨在限制感染患者中病毒复制的有吸引力的药物靶点。
Coronaviruses, like severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), encode a nucleotidyl transferase in the N-terminal (NiRAN) domain of thenonstructuralprotein (nsp) 12 protein within the RNA dependent RNA polymerase. Here we show the detection of guanosine monophosphate (GMP) and uridine monophosphate-modified amino acids in nidovirus proteins using heavy isotope-assisted mass spectrometry (MS) and MS/MS peptide sequencing. We identified lysine-143 in the equine arteritis virus (EAV) protein, nsp7, as a primary site of in vitro GMP attachment via a phosphoramide bond. In SARS-CoV-2 replicase proteins, we demonstrate nsp12-mediated nucleotidylation of nsp7 lysine-2. Our results demonstrate new strategies for detecting GMP-peptide linkages that can be adapted for higher throughput screening using mass spectrometric technologies. These data are expected to be important for a rapid and timely characterization of a new enzymatic activity in SARS-CoV-2 that may be an attractive drug target aimed at limiting viral replication in infected patients.