PDF(Pubmed)
Abstract：
The outbreak of coronavirus disease 2019 (COVID-19) has sparked an urgent demand for advanced diagnosis and vaccination worldwide. The discovery of high-affinity ligands is of great significance for vaccine and diagnostic reagent manufacturing. Targeting the receptor binding domain (RBD) from the spike protein of severe acute respiratory syndrome-coronavirus 2, an interface at the outer surface of helices on the Z domain from protein A was introduced to construct a virtual library for the screening of ZRBD affibody ligands. Molecular docking was performed using HADDOCK software, and three potential ZRBD affibodies, ZRBD-02, ZRBD-04, and ZRBD-07, were obtained. Molecular dynamics (MD) simulation verified that the binding of ZRBD affibodies to RBD was driven by electrostatic interactions. Per-residue free energy decomposition analysis further substantiated that four residues with negative-charge characteristics on helix α1 of the Z domain participated in this process. Binding affinity analysis by microscale thermophoresis showed that ZRBD affibodies had high affinity for RBD binding, and the lowest dissociation constant was 36.3 nmol/L for ZRBD-07 among the three potential ZRBD affibodies. Herein, ZRBD-02 and ZRBD-07 affibodies were selected for chromatographic verifications after being coupled to thiol-activated Sepharose 6 Fast Flow (SepFF) gel. Chromatographic experiments showed that RBD could bind on both ZRBD SepFF gels and was eluted by 0.1 mol/L NaOH. Moreover, the ZRBD-07 SepFF gel had a higher affinity for RBD. This research provided a new idea for the design of affibody ligands and validated the potential of affibody ligands in the application of RBD purification from complex feedstock.
摘要：
2019年冠状病毒病（COVID-19）的爆发引发了全球对高级诊断和疫苗接种的迫切需求。高亲和力配体的发现对疫苗和诊断试剂的生产具有重要意义。针对严重急性呼吸综合征-冠状病毒2的刺突蛋白的受体结合域（RBD），引入蛋白A的Z域螺旋外表面的界面，构建了用于筛选ZRBD亲和体配体的虚拟文库。使用HADDOCK软件进行分子对接，和三个潜在的ZRBD成员，获得ZRBD-02、ZRBD-04和ZRBD-07。分子动力学（MD）模拟验证了ZRBD亲和体与RBD的结合是由静电相互作用驱动的。每个残基的自由能分解分析进一步证实了Z结构域的螺旋α1上具有负电荷特征的四个残基参与了该过程。通过微尺度热电泳的结合亲和力分析显示ZRBD亲和体对RBD结合具有高亲和力，在三个潜在的ZRBD亲体中，ZRBD-07的最低解离常数为36.3nmol/L。在这里，在与硫醇活化的Sepharose6快速流动(SepFF)凝胶偶联后,选择ZRBD-02和ZRBD-07亲和体用于色谱验证。色谱实验表明，RBD可以在两种ZRBDSepFF凝胶上结合，并用0.1mol/L的NaOH洗脱。此外，ZRBD-07SepFF凝胶对RBD具有更高的亲和力。该研究为配合体配体的设计提供了新思路，并验证了配合体配体在复杂原料RBD纯化中的应用潜力。
