Computational electrostatic engineering of nanobodies for enhanced SARS-CoV-2 receptor binding domain recognition

This study presents a novel computational approach for engineering nanobodies (Nbs) for improved interaction with receptor-binding domain (RBD) of the SARS-CoV-2 spike protein. Using Protein Structure Reliability reports, RBD (7VYR_R) was selected and refined for subsequent Nb-RBD interactions. By leveraging electrostatic complementarity (EC) analysis, we engineered and characterized five Electrostatically Complementary Nbs (ECSb1-ECSb5) based on the CeVICA library's SR6c3 Nb. Through targeted modifications in the complementarity-determining regions (CDR) and framework regions (FR), we optimized electrostatic interactions to improve binding affinity and specificity. The engineered Nbs (ECSb3, ECSb4, and ECSb5) demonstrated high binding specificity for AS3, CA1, and CA2 epitopes. Interestingly, ECSb1 and ECSb2 selectively engaged with AS3 and CA1 instead of AS1 and AS2, respectively, due to a preference for residues that conferred superior binding complementarities. Furthermore, ECSbs significantly outperformed SR6c3 Nb in MM/GBSA results, notably, ECSb4 and ECSb3 exhibited superior binding free energies of -182.58 kcal.mol^-1 and -119.07 kcal.mol^-1, respectively, compared to SR6c3 (-105.50 kcal.mol^-1). ECSbs exhibited significantly higher thermostability (100.4-148.3 kcal·mol⁻¹) compared to SR6c3 (62.6 kcal·mol⁻¹). Similarly, enhanced electrostatic complementarity was also observed for ECSb4-RBD and ECSb3-RBD (0.305 and 0.390, respectively) relative to SR6c3-RBD (0.233). Surface analyses confirmed optimized electrostatic patches and reduced aggregation propensity in the engineered Nb. This integrated EC and structural engineering approach successfully developed engineered Nbs with enhanced binding specificity, increased thermostability, and reduced aggregation, laying the groundwork for novel therapeutic applications targeting the SARS-CoV-2 spike protein.