Pseudosymmetric hetero-oligomers with three or more unique subunits with overall structural (but not sequence) symmetry play key roles in biology, and systematic approaches for generating such proteins de novo would provide new routes to controlling cell signaling and designing complex protein materials. However, the de novo design of protein hetero-oligomers with three or more distinct chains with nearly identical structures is a challenging unsolved problem because it requires the accurate design of multiple protein-protein interfaces simultaneously. Here, we describe a divide-and-conquer approach that breaks the multiple-interface design challenge into a set of more tractable symmetric single-interface redesign tasks, followed by structural recombination of the validated homo-oligomers into pseudosymmetric hetero-oligomers. Starting from de novo designed circular homo-oligomers composed of 9 or 24 tandemly repeated units, we redesigned the inter-subunit interfaces to generate 19 new homo-oligomers and structurally recombined them to make 24 new hetero-oligomers, including ABC heterotrimers, A2B2 heterotetramers, and A3B3 and A2B2C2 heterohexamers which assemble with high structural specificity. The symmetric homo-oligomers and pseudosymmetric hetero-oligomers generated for each system have identical or nearly identical backbones, and hence are ideal building blocks for generating and functionalizing larger symmetric and pseudosymmetric assemblies.

具有三个或更多具有整体结构（但不是序列）对称性的独特亚基的伪对称异源寡聚体在生物学中起着关键作用，从头生成此类蛋白质的系统方法将为控制细胞信号传导和设计复杂蛋白质材料提供新途径。然而，具有三个或更多不同链且结构几乎相同的蛋白质异源寡聚体的从头设计是一个具有挑战性的未解决的问题，因为它需要同时准确设计多个蛋白质-蛋白质界面。在这里，我们描述了一种分而治之的方法，该方法将多界面设计挑战分解为一组更易于处理的对称单界面重新设计任务，然后将经过验证的同源寡聚体结构重组为伪对称异源寡聚体。从从 9 个或 24 个串联重复单元组成的循环同源寡聚体设计开始，我们重新设计了亚基间界面以生成 19 个新的同源寡聚体，并将它们在结构上重新组合以生成 24 个新的异源寡聚体，包括 ABC 异源三聚体、A2B2 异源四聚体以及 A3B3 和 A2B2C2 异源六聚体，它们以高度的结构特异性组装。为每个系统生成的对称同源寡聚体和伪对称异源寡聚体具有相同或几乎相同的主链，因此是生成和功能化更大的对称和伪对称组装体的理想构建模块。