Biophysical characterization of SARS-CoV-2 spike protein - receptor interactions

项目摘要/摘要 当前的冠状病毒病大流行-2019年(新冠肺炎)在世界各地产生了毁灭性的影响。 引起新冠肺炎的SARS-CoV-2病毒为了进入人类宿主细胞，使用其表面尖峰蛋白(S) 附着在宿主细胞表面受体上。除了最著名的受体ACE2外，还有许多细胞表面蛋白， 包括CD147NRP1和DC-SIGN/L-SIGN，已被报道与S蛋白结合，并与 调解SARS-CoV-2进入。始终如一地，我们的初步研究使用单分子力谱 结果表明，CD147、Nrp1和L-Sign能与SARS-CoV-2 S蛋白结合，其亲和力与 ACE2。因此，可能的多受体利用至少部分地解释了广泛的组织 SARS-CoV-2感染的嗜性与全身并发症然而，仍然令人困惑的是，S蛋白是如何 能够以高亲和力与这些结构不同的分子结合。此外，我们的全原子结构模型 资料显示，S蛋白表面大部分被多糖覆盖，只有当S蛋白的受体 结合结构域(RBD)处于“上位”，可以在不受糖链干扰的情况下与受体结合。因此，我们 假设S蛋白上未被多糖覆盖的有限区域，包括处于向上位置的RBD， 以及S1/S2连接区，可能负责与所有受体结合。在拟议的工作中， 我们将使用单病毒测力谱的组合方法系统地检验这一假设，所有- 原子分子模拟和模拟以及假病毒内化/进入试验。此外，自SARS以来- CoV-2有两条进入途径(病毒-宿主膜直接融合或内吞/巨噬细胞内化 随后是内体进入)，这些受体所介导的确切进入途径尚不清楚。这个 拟议中的研究将确定是否每一次个人互动都更容易中介直接病毒宿主 膜融合或病毒内吞/巨噬细胞内化。将追求两个具体目标：1) 研究S蛋白与宿主细胞膜受体的相互作用及2)确定其结构基础 S蛋白的广泛受体识别。这项研究将阐明其背后的结构和生物物理机制 S蛋白的受体识别与利用。这项工作的成功完成将使我们能够确定新的 新冠肺炎全身并发症的抗病毒治疗靶点。

PROJECT SUMMARY/ABSTRACT The current pandemic of Coronavirus Disease-2019 (COVID-19) has had devastating impacts across the world. In order to enter human host cells, SARS-CoV-2, the virus causing COVID-19, uses its surface spike (S) protein to attach to host cell surface receptors. Besides the best-known receptor, ACE2, a number of cell surface proteins, including CD147, neuropilin-1 (NRP1) and DC-SIGN/L-SIGN, have been reported to bind to S protein and mediate SARS-CoV-2 entry. Consistently, our preliminary studies using single-molecule force spectroscopy show that CD147, NRP1 and L-SIGN can bind to the SARS-CoV-2 S protein with comparable affinities to those of ACE2. Therefore, the possible multiple receptor utilization could, at least partially, explain the broad tissue tropism and systemic complications of SARS-CoV-2 infection. However, it remains puzzling how the S protein can bind to these structurally diverse molecules with high affinity. In addition, our all-atom structural modeling data shows that most of the S protein surface is covered by glycans, and only when the S protein’s receptor binding domain (RBD) is in the “up position” can it bind to a receptor without glycan interference. Therefore, we hypothesize that limited regions on the S protein that are not covered by glycans, including the RBD in up position, as well as the S1/S2 junctional region, may be responsible for binding all the receptors. In the proposed work, we will systematically test the hypothesis using combined approaches of single-virus force spectroscopy, all- atom molecular modeling and simulation and pseudovirus internalization/entry assays. Moreover, since SARS- CoV-2 has two entry routes (direct viral-host membrane fusion or endocytic/macropinocytic internalization followed by endosomal entry), the exact entry pathways that these receptors mediate are not yet clear. The proposed research will determine whether every individual interaction is more prone to mediate direct viral-host membrane fusion or viral endocytic/macropinocytic internalization. Two specific aims will be pursued: 1) to characterize S protein interactions with host cell membrane receptors and 2) to determine the structural basis of S protein’s broad receptor recognition. The study will elucidate the structural and biophysical mechanisms behind S protein’s receptor recognition and utilization. Successful completion of this work will allow us to identify new targets for antiviral therapies to treat the systemic complications of COVID-19.