Protein tertiary structure mimetics are valuable tools to target large protein–protein interaction interfaces. Here, we demonstrate a strategy for designing dimeric helix-hairpin motifs from a previously reported three-helix-bundle miniprotein that targets the receptor-binding domain (RBD) of severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2). Through truncation of the third helix and optimization of the interhelical loop residues of the miniprotein, we developed a thermostable dimeric helix-hairpin. The dimeric four-helix bundle competes with the human angiotensin-converting enzyme 2 (ACE2) in binding to RBD with 2:2 stoichiometry. Cryogenic-electron microscopy revealed the formation of dimeric spike ectodomain trimer by the four-helix bundle, where all the three RBDs from either spike protein are attached head-to-head in an open conformation, revealing a novel mechanism for virus neutralization. The proteomimetic protects hamsters from high dose viral challenge with replicative SARS-CoV-2 viruses, demonstrating the promise of this class of peptides that inhibit protein–protein interaction through target dimerization.

蛋白质三级结构模拟物是针对大型蛋白质-蛋白质相互作用界面的有价值的工具。在这里，我们展示了一种从先前报道的针对严重急性呼吸综合征冠状病毒 2 (SARS-CoV-2) 的受体结合域 (RBD) 的三螺旋束微型蛋白设计二聚螺旋-发夹基序的策略。通过截断第三螺旋和优化小蛋白的螺旋间环残基，我们开发了一种热稳定的二聚体螺旋-发夹。二聚四螺旋束与人血管紧张素转换酶 2 (ACE2) 竞争以 2:2 的化学计量与 RBD 结合。低温电子显微镜显示由四螺旋束形成二聚体刺突胞外域三聚体，其中任何一种刺突蛋白的所有三个 RBD 都以开放构象头对头连接，揭示了一种新的病毒中和机制。蛋白质模拟物可保护仓鼠免受复制性 SARS-CoV-2 病毒的高剂量病毒攻击，证明了这类肽通过靶点二聚化抑制蛋白质-蛋白质相互作用的前景。