PDF(Pubmed)
Abstract：
The development of methods able to modulate the binding affinity between proteins and peptides is of paramount biotechnological interest in view of a vast range of applications that imply designed polypeptides capable to impair or favour Protein-Protein Interactions. Here, we applied a peptide design algorithm based on shape complementarity optimization and electrostatic compatibility and provided the first experimental in vitro proof of the efficacy of the design algorithm. Focusing on the interaction between the SARS-CoV-2 Spike Receptor-Binding Domain (RBD) and the human angiotensin-converting enzyme 2 (ACE2) receptor, we extracted a 23-residues long peptide that structurally mimics the major interacting portion of the ACE2 receptor and designed in silico five mutants of such a peptide with a modulated affinity. Remarkably, experimental KD measurements, conducted using biolayer interferometry, matched the in silico predictions. Moreover, we investigated the molecular determinants that govern the variation in binding affinity through molecular dynamics simulation, by identifying the mechanisms driving the different values of binding affinity at a single residue level. Finally, the peptide sequence with the highest affinity, in comparison with the wild type peptide, was expressed as a fusion protein with human H ferritin (HFt) 24-mer. Solution measurements performed on the latter constructs confirmed that peptides still exhibited the expected trend, thereby enhancing their efficacy in RBD binding. Altogether, these results indicate the high potentiality of this general method in developing potent high-affinity vectors for hindering/enhancing protein-protein associations.
摘要：
鉴于大量应用暗示设计的多肽能够削弱或有利于蛋白质-蛋白质相互作用,开发能够调节蛋白质和肽之间的结合亲和力的方法具有最重要的生物技术兴趣。这里,我们应用了一种基于形状互补优化和静电相容性的肽设计算法，并首次在体外实验证明了设计算法的有效性。着眼于SARS-CoV-2尖峰受体结合域（RBD）与人血管紧张素转换酶2（ACE2）受体之间的相互作用，我们提取了23个残基长的肽，该肽在结构上模拟了ACE2受体的主要相互作用部分，并在计算机上设计了具有调节亲和力的这种肽的五个突变体。值得注意的是,实验性KD测量，使用生物层干涉法进行，与计算机预测相符。此外，我们通过分子动力学模拟研究了控制结合亲和力变化的分子决定因素，通过确定在单个残基水平上驱动不同结合亲和力值的机制。最后,具有最高亲和力的肽序列，与野生型肽相比，表达为与人H铁蛋白(HFt)24-mer的融合蛋白。对后一种构建体进行的溶液测量证实，肽仍然表现出预期的趋势。从而增强它们在RBD结合中的功效。总之,这些结果表明，这种通用方法在开发阻碍/增强蛋白质-蛋白质缔合的有效高亲和力载体方面具有很高的潜力。
