Stabilization of the SARS-CoV-2 receptor binding domain by protein core redesign and deep mutational scanning
Stabilization of the SARS-CoV-2 receptor binding domain by protein core redesign and deep mutational scanning
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DOI：
10.1093/protein/gzac002
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发表时间：
2022-02-17
影响因子：
2.4
通讯作者：
Whitehead, Timothy A.
中科院分区：
文献类型：
--
作者：
Leonard, Alison C.;Weinstein, Jonathan J.;Whitehead, Timothy A.
稳定抗原蛋白作为疫苗免疫原或诊断试剂是蛋白质工程和设计的严格情况，因为外表面必须保持在多个不同表位处被受体和抗原特异性抗体识别。这是一个挑战，因为增强稳定性的突变必须集中在蛋白质核心上，而成功的计算稳定算法通常在面向溶剂的位置选择突变。在这项研究中，我们报告了SARS-CoV-2武汉Hu-1刺突受体结合结构域的稳定性，使用深度突变扫描和计算设计相结合，包括FuncLib算法。我们最成功的设计编码I358 F、Y365 W、T430 I和I513 L受体结合结构域突变，保持受体ACE 2和一组不同抗受体结合结构域单克隆抗体的识别，使用热位移测定，比原始受体结合结构域热稳定1至2 ℃，并且对糜蛋白酶和嗜热菌蛋白酶的蛋白水解敏感性低于原始受体结合结构域。我们的方法可以应用于计算稳定的广泛的蛋白质，而不需要详细的知识的活性位点或结合表位。我们设想，这种策略可能是特别强大的情况下，当有多个或未知的结合位点。
Stabilizing antigenic proteins as vaccine immunogens or diagnostic reagents is a stringent case of protein engineering and design as the exterior surface must maintain recognition by receptor(s) and antigen-specific antibodies at multiple distinct epitopes. This is a challenge, as stability enhancing mutations must be focused on the protein core, whereas successful computational stabilization algorithms typically select mutations at solvent-facing positions. In this study, we report the stabilization of SARS-CoV-2 Wuhan Hu-1 Spike receptor binding domain using a combination of deep mutational scanning and computational design, including the FuncLib algorithm. Our most successful design encodes I358F, Y365W, T430I, and I513L receptor binding domain mutations, maintains recognition by the receptor ACE2 and a panel of different anti-receptor binding domain monoclonal antibodies, is between 1 and 2 degrees C more thermally stable than the original receptor binding domain using a thermal shift assay, and is less proteolytically sensitive to chymotrypsin and thermolysin than the original receptor binding domain. Our approach could be applied to the computational stabilization of a wide range of proteins without requiring detailed knowledge of active sites or binding epitopes. We envision that this strategy may be particularly powerful for cases when there are multiple or unknown binding sites.