Mani Natesan, Suresh Raghavendran, Chakraborty Srirupa
Department of Chemical Engineering, Northeastern University, Boston, MA 02115; Department of Chemistry and Chemical Biology, Northeastern University, Boston, MA 02115.
bioRxiv. 2025 Mar 14:2025.03.12.642945. doi: 10.1101/2025.03.12.642945.
The SARS-CoV-2 Spike protein is the primary target for vaccine design, with immunogens typically engineered to enhance stability by introducing proline mutations (2P) and mutating or deleting the Furin Cleavage Site (FCS). While these modifications improve structural integrity, studies suggest that furin cleavage can play a functional role in Spike protein dynamics, potentially enhancing ACE2 receptor binding. However, the impact of this cleavage on the unbound form of the Spike protein remains unclear. In this study, we use extensive all-atom molecular dynamics (MD) simulations to compare the structural and dynamic properties of cleaved and uncleaved Spike proteins in their pre-fusion, unbound state. Our results show that Furin cleavage significantly alters allosteric communication within the protein, increasing correlated motions between the Receptor Binding Domain (RBD) and N-terminal Domain (NTD), which may facilitate receptor engagement. Principal Component Analysis (PCA) reveals that the cleaved and uncleaved Spike proteins sample distinct conformational landscapes, with the cleaved form displaying enhanced flexibility and a broader range of RBD tilt angles. Additionally, Furin cleavage primes the S2 subunit by expanding the central helix, potentially influencing the transition to the post-fusion state. Glycan clustering patterns further suggest an adaptive structural response to cleavage, particularly in the NTD and RBD regions. These findings highlight the potential functional consequences of FCS deletion in immunogen design and underscore the importance of considering the native cleavage state in vaccine and therapeutic development.
严重急性呼吸综合征冠状病毒2（SARS-CoV-2）刺突蛋白是疫苗设计的主要靶点，免疫原通常经过工程改造，通过引入脯氨酸突变（2P）以及突变或删除弗林蛋白酶切割位点（FCS）来提高稳定性。虽然这些修饰改善了结构完整性，但研究表明弗林蛋白酶切割在刺突蛋白动力学中可能发挥功能性作用，有可能增强血管紧张素转换酶2（ACE2）受体结合。然而，这种切割对刺突蛋白未结合形式的影响仍不清楚。在本研究中，我们使用广泛的全原子分子动力学（MD）模拟来比较切割和未切割的刺突蛋白在融合前未结合状态下的结构和动力学特性。我们的结果表明，弗林蛋白酶切割显著改变了蛋白质内的别构通讯，增加了受体结合结构域（RBD）和N端结构域（NTD）之间的相关运动，这可能有助于受体结合。主成分分析（PCA）显示，切割和未切割的刺突蛋白采样不同的构象景观，切割形式显示出增强的灵活性和更广泛的RBD倾斜角度范围。此外，弗林蛋白酶切割通过扩展中央螺旋使S2亚基预激活，可能影响向后融合状态的转变。聚糖聚类模式进一步表明对切割的适应性结构反应，特别是在NTD和RBD区域。这些发现突出了FCS缺失在免疫原设计中的潜在功能后果，并强调了在疫苗和治疗开发中考虑天然切割状态的重要性。
