Camelid single-domain antibodies, also known as nanobodies, can be readily isolated from naïve libraries for specific targets but often bind too weakly to their targets to be immediately useful. Laboratory-based genetic engineering methods to enhance their affinity, termed maturation, can deliver useful reagents for different areas of biology and potentially medicine. Using the receptor binding domain (RBD) of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein and a naïve library, we generated closely related nanobodies with micromolar to nanomolar binding affinities. By analyzing the structure–activity relationship using X-ray crystallography, cryoelectron microscopy, and biophysical methods, we observed that higher conformational entropy losses in the formation of the spike protein–nanobody complex are associated with tighter binding. To investigate this, we generated structural ensembles of the different complexes from electron microscopy maps and correlated the conformational fluctuations with binding affinity. This insight guided the engineering of a nanobody with improved affinity for the spike protein.

中文翻译：

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纳米抗体 SARS-CoV-2 刺突蛋白复合物的结合亲和力与构象熵之间的相关性

骆驼单域抗体，也称为纳米抗体，可以很容易地从特定靶标的初始文库中分离出来，但通常与其靶标结合太弱，无法立即使用。基于实验室的基因工程方法可以增强其亲和力（称为成熟），可以为生物学和潜在医学的不同领域提供有用的试剂。利用严重急性呼吸综合征冠状病毒 2 (SARS-CoV-2) 刺突蛋白的受体结合域 (RBD) 和初始文库，我们生成了具有微摩尔至纳摩尔结合亲和力的密切相关的纳米抗体。通过使用X射线晶体学、冷冻电子显微镜和生物物理方法分析结构-活性关系，我们观察到刺突蛋白-纳米抗体复合物形成过程中较高的构象熵损失与更紧密的结合有关。为了研究这一点，我们从电子显微镜图上生成了不同复合物的结构整体，并将构象波动与结合亲和力相关联。这一见解指导了对刺突蛋白具有更高亲和力的纳米抗体的工程设计。