期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2022
卷号：119
期号：31
DOI：10.1073/pnas.2205412119
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：Significance
Understanding the structural principles that determine the binding affinity of nanobodies to the spike protein of severe acute respiratory syndrome coronavirus 2 has been difficult. We analyzed electron microscopy maps of nanobody-spike complexes and quantified the conformational entropy of binding. This informed the design of an engineered nanobody with improved binding to the spike protein. This result offers a guiding principle for the rational maturation of nanobodies directed against the spike. High-binding potency nanobodies have been shown to be effective in animal models; thus, this technology could have application in future pandemics.
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.
