Iqbal Zafar, Asim Muhammad, Khan Umair Ahmad, Sultan Neelam, Ali Irfan
Central Laboratories, King Faisal University, Al Hofuf, Saudi Arabia.
Centre of Agricultural Biochemistry and Biotechnology (CABB), University of Agriculture, Faisalabad, Pakistan.
Front Mol Biosci. 2025 Mar 10;12:1512788. doi: 10.3389/fmolb.2025.1512788. eCollection 2025.
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 and -119.07 kcal.mol, respectively, compared to SR6c3 (-105.50 kcal.mol). ECSbs exhibited significantly higher thermostability (100.4-148.3 kcal·mol⁻) compared to SR6c3 (62.6 kcal·mol⁻). 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.
本研究提出了一种新颖的计算方法，用于设计纳米抗体（Nb），以改善其与严重急性呼吸综合征冠状病毒2（SARS-CoV-2）刺突蛋白受体结合域（RBD）的相互作用。利用蛋白质结构可靠性报告，选择并优化了RBD（7VYR_R），用于后续的Nb-RBD相互作用研究。通过利用静电互补性（EC）分析，我们基于CeVICA文库的SR6c3 Nb设计并表征了5种静电互补纳米抗体（ECSb1-ECSb5）。通过对互补决定区（CDR）和框架区（FR）进行有针对性的修饰，我们优化了静电相互作用，以提高结合亲和力和特异性。工程化的纳米抗体（ECSb3、ECSb4和ECSb5）对AS3、CA1和CA2表位表现出高结合特异性。有趣的是，由于对赋予优异结合互补性的残基的偏好，ECSb1和ECSb2分别选择性地与AS3和CA1结合，而不是与AS1和AS2结合。此外，在MM/GBSA结果中，ECSbs显著优于SR6c3 Nb，值得注意的是，与SR6c3（-105.50 kcal.mol）相比，ECSb4和ECSb3分别表现出-182.58 kcal.mol和-119.07 kcal.mol的优异结合自由能。与SR6c3（62.6 kcal·mol⁻）相比，ECSbs表现出显著更高的热稳定性（100.4 - 148.3 kcal·mol⁻）。同样，相对于SR6c3-RBD（0.233），ECSb4-RBD和ECSb3-RBD的静电互补性也增强（分别为0.305和0.390）。表面分析证实了工程化纳米抗体中静电斑块的优化和聚集倾向的降低。这种整合的EC和结构工程方法成功开发出了具有增强结合特异性、提高热稳定性和减少聚集的工程化纳米抗体，为针对SARS-CoV-2刺突蛋白的新型治疗应用奠定了基础。
