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 Z(RBD) affibody ligands. Molecular docking was performed using HADDOCK software, and three potential Z(RBD) affibodies, Z(RBD)-02, Z(RBD)-04, and Z(RBD)-07, were obtained. Molecular dynamics (MD) simulation verified that the binding of Z(RBD) 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 Z(RBD) affibodies had high affinity for RBD binding, and the lowest dissociation constant was 36.3 nmol/L for Z(RBD)-07 among the three potential Z(RBD) affibodies. Herein, Z(RBD)-02 and Z(RBD)-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 Z(RBD) SepFF gels and was eluted by 0.1 mol/L NaOH. Moreover, the Z(RBD)-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.