NHC Key Laboratory of Systems Biology of Pathogens, Institute of Pathogen Biology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China.
Sanming Project of Medicine in Shenzhen, National Clinical Research Center for Infectious Diseases, Shenzhen Third People's Hospital, Southern University of Science and Technology, Shenzhen, China.
Nat Commun. 2021 May 14;12(1):2843. doi: 10.1038/s41467-021-23118-8.
Although the accessory proteins are considered non-essential for coronavirus replication, accumulating evidences demonstrate they are critical to virus-host interaction and pathogenesis. Orf9b is a unique accessory protein of SARS-CoV-2 and SARS-CoV. It is implicated in immune evasion by targeting mitochondria, where it associates with the versatile adapter TOM70. Here, we determined the crystal structure of SARS-CoV-2 orf9b in complex with the cytosolic segment of human TOM70 to 2.2 Å. A central portion of orf9b occupies the deep pocket in the TOM70 C-terminal domain (CTD) and adopts a helical conformation strikingly different from the β-sheet-rich structure of the orf9b homodimer. Interactions between orf9b and TOM70 CTD are primarily hydrophobic and distinct from the electrostatic interaction between the heat shock protein 90 (Hsp90) EEVD motif and the TOM70 N-terminal domain (NTD). Using isothermal titration calorimetry (ITC), we demonstrated that the orf9b dimer does not bind TOM70, but a synthetic peptide harboring a segment of orf9b (denoted C-peptide) binds TOM70 with nanomolar K. While the interaction between C-peptide and TOM70 CTD is an endothermic process, the interaction between Hsp90 EEVD and TOM70 NTD is exothermic, which underscores the distinct binding mechanisms at NTD and CTD pockets. Strikingly, the binding affinity of Hsp90 EEVD motif to TOM70 NTD is reduced by ~29-fold when orf9b occupies the pocket of TOM70 CTD, supporting the hypothesis that orf9b allosterically inhibits the Hsp90/TOM70 interaction. Our findings shed light on the mechanism underlying SARS-CoV-2 orf9b mediated suppression of interferon responses.
虽然辅助蛋白被认为对冠状病毒复制不是必需的，但越来越多的证据表明它们对病毒-宿主相互作用和发病机制至关重要。Orf9b 是 SARS-CoV-2 和 SARS-CoV 的独特辅助蛋白。它通过靶向线粒体来逃避免疫，在那里它与多功能适配器 TOM70 相关。在这里，我们确定了 SARS-CoV-2 orf9b 与人类 TOM70 胞质段复合物的晶体结构，分辨率为 2.2Å。orf9b 的中心部分占据了 TOM70 C 端结构域 (CTD) 的深口袋，并采用了与 orf9b 同源二聚体富含 β 片层结构明显不同的螺旋构象。orf9b 与 TOM70 CTD 之间的相互作用主要是疏水的，与热休克蛋白 90 (Hsp90) EEVD 基序与 TOM70 N 端结构域 (NTD) 之间的静电相互作用不同。使用等温滴定量热法 (ITC)，我们证明了二聚体 orf9b 不结合 TOM70，但含有 orf9b 片段的合成肽（称为 C 肽）以纳摩尔亲和力结合 TOM70。虽然 C 肽与 TOM70 CTD 之间的相互作用是一个吸热过程，但 Hsp90 EEVD 与 TOM70 NTD 之间的相互作用是放热的，这凸显了 NTD 和 CTD 口袋之间的不同结合机制。引人注目的是，当 orf9b 占据 TOM70 CTD 的口袋时，Hsp90 EEVD 基序与 TOM70 NTD 的结合亲和力降低了约 29 倍，支持了 orf9b 变构抑制 Hsp90/TOM70 相互作用的假说。我们的研究结果揭示了 SARS-CoV-2 orf9b 介导的干扰素反应抑制的机制。
