De novo protein design has enabled the creation of new protein structures. However, the design of functional proteins has proved challenging, in part due to the difficulty of transplanting structurally complex functional sites to available protein structures. Here, we used a bottom-up approach to build de novo proteins tailored to accommodate structurally complex functional motifs. We applied the bottom-up strategy to successfully design five folds for four distinct binding motifs, including a bifunctionalized protein with two motifs. Crystal structures confirmed the atomic-level accuracy of the computational designs. These de novo proteins were functional as components of biosensors to monitor antibody responses and as orthogonal ligands to modulate synthetic signaling receptors in engineered mammalian cells. Our work demonstrates the potential of bottom-up approaches to accommodate complex structural motifs, which will be essential to endow de novo proteins with elaborate biochemical functions, such as molecular recognition or catalysis.

从头蛋白质设计使新蛋白质结构的产生成为可能。然而，功能性蛋白质的设计已被证明具有挑战性，部分原因是难以将结构复杂的功能位点移植到可用的蛋白质结构中。在这里，我们使用自下而上的方法来构建为适应结构复杂的功能基序而量身定制的从头蛋白质。我们应用自下而上的策略成功地为四个不同的结合基序设计了五个折叠，包括具有两个基序的双功能化蛋白质。晶体结构证实了计算设计的原子级精度。这些从头合成的蛋白质可作为生物传感器的成分来监测抗体反应，并作为正交配体来调节工程哺乳动物细胞中的合成信号受体。