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三螺旋束小蛋白中设计二聚体螺旋发夹基序的策略，该小蛋白靶向严重急性呼吸综合征冠状病毒2（SARS-CoV-2）的受体结合域（RBD）。通过第三螺旋的截断和小蛋白螺旋间环残基的优化，我们开发了一种热稳定的二聚体螺旋发夹。二聚体四螺旋束与人血管紧张素转换酶 2 (ACE2) 竞争以 2:2 化学计量与 RBD 结合。低温电子显微镜揭示了四螺旋束形成二聚刺突胞外域三聚体，其中任一刺突蛋白的所有三个 RBD 以开放构象头对头连接，揭示了病毒中和的新机制。这种蛋白质模拟物可以保护仓鼠免受复制性 SARS-CoV-2 病毒的高剂量病毒攻击，这证明了此类肽通过目标二聚化抑制蛋白质-蛋白质相互作用的前景。