Eunice Kennedy Shriver National Institute of Child Health and Human Development, NIH, Bethesda, MD 20892.
Department of Chemistry, The Pennsylvania State University, University Park, PA 16802.
Proc Natl Acad Sci U S A. 2023 Aug 15;120(33):e2303860120. doi: 10.1073/pnas.2303860120. Epub 2023 Aug 8.
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19, uses an RNA-dependent RNA polymerase along with several accessory factors to replicate its genome and transcribe its genes. Nonstructural protein (nsp) 13 is a helicase required for viral replication. Here, we found that nsp13 ligates iron, in addition to zinc, when purified anoxically. Using inductively coupled plasma mass spectrometry, UV-visible absorption, EPR, and Mössbauer spectroscopies, we characterized nsp13 as an iron-sulfur (Fe-S) protein that ligates an FeS cluster in the treble-clef metal-binding site of its zinc-binding domain. The Fe-S cluster in nsp13 modulates both its binding to the template RNA and its unwinding activity. Exposure of the protein to the stable nitroxide TEMPOL oxidizes and degrades the cluster and drastically diminishes unwinding activity. Thus, optimal function of nsp13 depends on a labile Fe-S cluster that is potentially targetable for COVID-19 treatment.
严重急性呼吸综合征冠状病毒 2（SARS-CoV-2）是 COVID-19 的病原体，它利用 RNA 依赖性 RNA 聚合酶和几种辅助因子来复制其基因组并转录其基因。非结构蛋白（nsp）13 是病毒复制所需的解旋酶。在这里，我们发现 nsp13 在缺氧条件下除了锌之外还可以结合铁。我们使用电感耦合等离子体质谱、紫外可见吸收、EPR 和 Mössbauer 光谱学，将 nsp13 鉴定为一种铁硫（Fe-S）蛋白，它在锌结合域的高音谱号金属结合位点结合一个 FeS 簇。nsp13 中的 Fe-S 簇调节其与模板 RNA 的结合及其解旋活性。将蛋白质暴露于稳定的氮氧自由基 TEMPOL 会氧化和降解该簇，并大大降低解旋活性。因此，nsp13 的最佳功能取决于一个不稳定的 Fe-S 簇，该簇可能成为 COVID-19 治疗的靶点。
