Singh Suruchi, Liu Yi, Burke Meghan, Rayaprolu Vamseedhar, Stein Stephen E, Hasan S Saif
Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore MD 21201, USA.
Mass Spectrometry Data Center, Biomolecular Measurement Division, National Institute of Standards and Technology, Gaithersburg MD 20899, USA.
J Struct Biol X. 2025 Feb 11;11:100123. doi: 10.1016/j.yjsbx.2025.100123. eCollection 2025 Jun.
The SARS-CoV-2 spike protein is synthesized in the endoplasmic reticulum of host cells, from where it undergoes export to the Golgi and the plasma membrane or retrieval from the Golgi to the endoplasmic reticulum. Elucidating the fundamental principles of this bidirectional secretion are pivotal to understanding virus assembly and designing the next generation of spike genetic vaccine with enhanced export properties. However, the widely used strategy of C-terminal affinity tagging of the spike cytosolic tail interferes with proper bidirectional trafficking. Hence, the structural and biophysical investigations of spike protein trafficking have been hindered by a lack of appropriate spike constructs. Here we describe a strategy for the internal tagging of the spike protein. Using sequence analyses and AlphaFold modeling, we identified a site down-stream of the signal sequence for the insertion of a twin-strep-tag, which facilitates purification of an ecto-domain construct from the extra-cellular medium of mammalian Expi293F cells. Mass spectrometry analyses show that the internal tag has minimal impact on -glycan modifications, which are pivotal for spike-host interactions. Single particle cryo-electron microscopy reconstructions of the spike ecto-domain reveal conformational states compatible for ACE2 receptor interactions, further solidifying the feasibility of the internal tagging strategy. Collectively, these results present a substantial advance towards reagent development for the investigations of spike protein trafficking during coronavirus infection and genetic vaccination.
严重急性呼吸综合征冠状病毒2（SARS-CoV-2）刺突蛋白在宿主细胞的内质网中合成，从内质网输出至高尔基体和质膜，或从高尔基体返回内质网。阐明这种双向分泌的基本原理对于理解病毒组装以及设计具有增强输出特性的下一代刺突基因疫苗至关重要。然而，广泛使用的对刺突蛋白胞质尾进行C端亲和标记的策略会干扰正常的双向运输。因此，缺乏合适的刺突蛋白构建体阻碍了对刺突蛋白运输的结构和生物物理研究。在此，我们描述了一种对刺突蛋白进行内部标记的策略。通过序列分析和AlphaFold建模，我们确定了信号序列下游的一个位点用于插入双Strep标签，这有助于从哺乳动物Expi293F细胞的细胞外培养基中纯化胞外域构建体。质谱分析表明，内部标签对N - 聚糖修饰的影响最小，而N - 聚糖修饰对于刺突蛋白与宿主的相互作用至关重要。刺突蛋白胞外域的单颗粒冷冻电子显微镜重建揭示了与血管紧张素转换酶2（ACE2）受体相互作用相容的构象状态，进一步证实了内部标记策略的可行性。总体而言，这些结果为开发用于研究冠状病毒感染期间刺突蛋白运输和基因疫苗接种的试剂取得了重大进展。
