Abiotic d-proteins that selectively bind to natural l-proteins have gained significant biotechnological interest. However, the underlying structural principles governing such heterochiral protein–protein interactions remain largely unknown. In this study, we present the de novo design of d-proteins consisting of 50–65 residues, aiming to target specific surface regions of l-proteins or l-peptides. Our designer d-protein binders exhibit nanomolar affinity toward an artificial l-peptide, as well as two naturally occurring proteins of therapeutic significance: the D5 domain of human tropomyosin receptor kinase A (TrkA) and human interleukin-6 (IL-6). Notably, these d-protein binders demonstrate high enantiomeric specificity and target specificity. In cell-based experiments, designer d-protein binders effectively inhibited the downstream signaling of TrkA and IL-6 with high potency. Moreover, these binders exhibited remarkable thermal stability and resistance to protease degradation. Crystal structure of the designed heterochiral d-protein–l-peptide complex, obtained at a resolution of 2.0 Å, closely resembled the design model, indicating that the computational method employed is highly accurate. Furthermore, the crystal structure provides valuable information regarding the interactions between helical l-peptides and d-proteins, particularly elucidating a novel mode of heterochiral helix–helix interactions. Leveraging the design of d-proteins specifically targeting l-peptides or l-proteins opens up avenues for systematic exploration of the mirror-image protein universe, paving the way for a diverse range of applications.

选择性地与天然 L 蛋白结合的非生物 d 蛋白已获得重要的生物技术兴趣。然而，控制这种异手性蛋白质-蛋白质相互作用的基本结构原理在很大程度上仍然未知。在这项研究中，我们提出了由 50-65 个残基组成的 d 蛋白的从头设计，旨在靶向 l 蛋白或 l 肽的特定表面区域。我们设计的 d 蛋白结合剂对人工 L 肽以及两种具有治疗意义的天然存在的蛋白质表现出纳摩尔亲和力：人原肌球蛋白受体激酶 A （TrkA） 和人白细胞介素-6 （IL-6） 的 D5 结构域。值得注意的是，这些 d 蛋白结合物表现出高对映体特异性和靶标特异性。在基于细胞的实验中，设计型 d 蛋白结合剂有效抑制 TrkA 和 IL-6 的下游信号传导，且高效。此外，这些结合剂表现出显著的热稳定性和抗蛋白酶降解性。以 2.0 Å 的分辨率获得的所设计的异手性 d-蛋白-l-肽复合物的晶体结构与设计模型非常相似，表明所采用的计算方法非常准确。此外，晶体结构提供了有关螺旋 L 肽和 d 蛋白之间相互作用的宝贵信息，特别是阐明了异手性螺旋-螺旋相互作用的新模式。 利用专门靶向 L 肽或 L 蛋白的 d 蛋白设计为系统探索镜像蛋白世界开辟了途径，为各种应用铺平了道路。