Quantitative Assessment of Energetic Contributions of Residues in a SARS-CoV-2 Viral Enzyme/Nanobody Interface
Quantitative Assessment of Energetic Contributions of Residues in a SARS-CoV-2 Viral Enzyme/Nanobody Interface
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DOI：
10.1021/acs.jcim.3c01933
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发表时间：
2024-03-09
影响因子：
5.6
通讯作者：
Vashisth，Harish
中科院分区：
文献类型：
--
作者：
Kumar，Amit;Vashisth，Harish
SARS-CoV-2（MPro）中高度保守的蛋白酶对于病毒复制至关重要，并且是设计新型抑制化合物的有吸引力的靶点。已知MPro在构象上是柔性的，并且已经在与新型纳米抗体（NB 2B 4）的复合物中稳定在延伸构象中，所述纳米抗体通过结合到变构位点来抑制酶的二聚化。然而，纳米抗体残留物稳定MPro/纳米抗体界面的能量贡献仍然没有得到解决。我们探测这些残基使用全原子MD模拟结合炼金术自由能计算通过研究物理残基-残基相互作用，并发现疏水和静电相互作用的作用，在稳定的复合物。具体地，我们通过突变分析发现，三个界面纳米抗体残基（Y 59、R106和L109）对纳米抗体的总体结合亲和力贡献显著，两个残基（L107和P110）贡献中等，两个残基（H112和T113）贡献最小。我们还发现，纳米抗体的亲和力可以通过电荷逆转突变（D 62 R）来增强，该突变改变了复合物中该残基的局部界面静电环境。这些发现在设计新型合成纳米抗体作为MPro的变构抑制剂方面是潜在有用的。
The highly conserved protease enzyme from SARS-CoV-2 (MPro) is crucial for viral replication and is an attractive target for the design of novel inhibitory compounds. MProis known to be conformationally flexible and has been stabilized in an extended conformation in a complex with a novel nanobody (NB2B4), which inhibits the dimerization of the enzyme via binding to an allosteric site. However, the energetic contributions of the nanobody residues stabilizing the MPro/nanobody interface remain unresolved. We probed these residues using all-atom MD simulations in combination with alchemical free energy calculations by studying the physical residue–residue interactions and discovered the role of hydrophobic and electrostatic interactions in stabilizing the complex. Specifically, we found via mutational analysis that three interfacial nanobody residues (Y59, R106, and L109) contributed significantly, two residues (L107 and P110) contributed moderately, and two residues (H112 and T113) contributed minimally to the overall binding affinity of the nanobody. We also discovered that the nanobody affinity could be enhanced via a charge-reversal mutation (D62R) that alters the local interfacial electrostatic environment of this residue in the complex. These findings are potentially useful in designing novel synthetic nanobodies as allosteric inhibitors of MPro.