Kensinger Adam H, Makowski Joseph A, Pellegrene Kendy A, Imperatore Joshua A, Cunningham Caylee L, Frye Caleb J, Lackey Patrick E, Mihailescu Mihaela Rita, Evanseck Jeffrey D
Department of Chemistry and Biochemistry and Center for Computational Sciences, Duquesne University, Pittsburgh, Pennsylvania15282, United States.
Department of Biochemistry and Chemistry, Westminster College, New Wilmington, Pennsylvania16172, United States.
ACS Phys Chem Au. 2022 Oct 4;3(1):30-43. doi: 10.1021/acsphyschemau.2c00032. eCollection 2023 Jan 25.
The functional role of the highly conserved stem-loop II motif (s2m) in SARS-CoV and SARS-CoV-2 in the viral lifecycle remains enigmatic and an intense area of research. Structure and dynamics of the s2m are key to establishing a structure-function connection, yet a full set of atomistic resolution coordinates is not available for SARS-CoV-2 s2m. Our work constructs three-dimensional coordinates consistent with NMR solution phase data for SARS-CoV-2 s2m and provides a comparative analysis with its counterpart SARS-CoV s2m. We employed initial coordinates based on PDB ID 1XJR for SARS-CoV s2m and two models for SARS-CoV-2 s2m: one based on 1XJR in which we introduced the mutations present in SARS-CoV-2 s2m and the second based on the available SARS-CoV-2 NMR NOE data supplemented with knowledge-based methods. For each of the three systems, 3.5 μs molecular dynamics simulations were used to sample the structure and dynamics, and principal component analysis (PCA) reduced the ensembles to hierarchal conformational substates for detailed analysis. Dilute solution simulations of SARS-CoV s2m demonstrate that the GNRA-like terminal pentaloop is rigidly defined by base stacking uniquely positioned for possible kissing dimer formation. However, the SARS-CoV-2 s2m simulation did not retain the reported crystallographic SARS-CoV motifs and the terminal loop expands to a highly dynamic "nonaloop." Increased flexibility and structural disorganization are observed for the larger terminal loop, where an entropic penalty is computed to explain the experimentally observed reduction in kissing complex formation. Overall, both SARS-CoV and SARS-CoV-2 s2m elements have a similarly pronounced L-shape due to different motif interactions. Our study establishes the atomistic three-dimensional structure and uncovers dynamic differences that arise from s2m sequence changes, which sets the stage for the interrogation of different mechanistic pathways of suspected biological function.
高度保守的茎环II基序（s2m）在严重急性呼吸综合征冠状病毒（SARS-CoV）和新冠病毒（SARS-CoV-2）病毒生命周期中的功能作用仍然不明，是一个热门研究领域。s2m的结构和动力学是建立结构-功能联系的关键，但尚无新冠病毒s2m的全套原子分辨率坐标。我们的工作构建了与新冠病毒s2m的核磁共振（NMR）溶液相数据一致的三维坐标，并与其对应的SARS-CoV s2m进行了比较分析。我们采用基于蛋白质数据银行（PDB）ID 1XJR的初始坐标来构建SARS-CoV s2m，并为新冠病毒s2m构建了两个模型：一个基于1XJR，其中引入了新冠病毒s2m中存在的突变；另一个基于可用的新冠病毒NMR核欧沃豪斯效应（NOE）数据，并辅以基于知识的方法。对于这三个系统中的每一个，均使用3.5微秒的分子动力学模拟来采样结构和动力学，主成分分析（PCA）将系综简化为分层构象亚态以进行详细分析。SARS-CoV s2m的稀溶液模拟表明，类似GNRA的末端五聚环由碱基堆积严格定义，其独特的位置可能形成亲吻二聚体。然而，新冠病毒s2m模拟并未保留所报道的SARS-CoV晶体基序，且末端环扩展为高度动态的“非环”。在较大的末端环中观察到了更大的灵活性和结构无序，计算了熵罚来解释实验观察到的亲吻复合物形成减少的现象。总体而言，由于不同的基序相互作用，SARS-CoV和新冠病毒的s2m元件都具有类似明显的L形。我们的研究建立了原子三维结构，揭示了由s2m序列变化引起的动态差异，这为探究可疑生物学功能的不同机制途径奠定了基础。
