Adams Chloe S, Kim Hyojin, Burtner Abigail E, Lee Dong Sun, Dobbins Craig, Criswell Cameron, Coventry Brian, Tran-Pearson Adri, Kim Ho Min, King Neil P
Institute for Protein Design, University of Washington, Seattle, WA, 98195, USA.
Department of Biochemistry, University of Washington, Seattle, WA, 98195, USA.
Nat Commun. 2025 Jan 31;16(1):1234. doi: 10.1038/s41467-025-56369-w.
Toll-like Receptor 3 (TLR3) is a pattern recognition receptor that initiates antiviral immune responses upon binding double-stranded RNA (dsRNA). Several nucleic acid-based TLR3 agonists have been explored clinically as vaccine adjuvants in cancer and infectious disease, but present substantial manufacturing and formulation challenges. Here, we use computational protein design to create novel miniproteins that bind to human TLR3 with nanomolar affinities. Cryo-EM structures of two minibinders in complex with TLR3 reveal that they bind the target as designed, although one partially unfolds due to steric competition with a nearby N-linked glycan. Multivalent forms of both minibinders induce NF-κB signaling in TLR3-expressing cell lines, demonstrating that they may have therapeutically relevant biological activity. Our work provides a foundation for the development of specific, stable, and easy-to-formulate protein-based agonists of TLRs and other pattern recognition receptors.
Toll样受体3（TLR3）是一种模式识别受体，在结合双链RNA（dsRNA）后启动抗病毒免疫反应。几种基于核酸的TLR3激动剂已在临床上作为癌症和传染病的疫苗佐剂进行探索，但目前存在重大的生产和制剂挑战。在此，我们利用计算蛋白质设计来创建与人类TLR3以纳摩尔亲和力结合的新型小蛋白。两种与TLR3复合的小结合剂的冷冻电镜结构表明，它们按设计结合靶标，尽管其中一种由于与附近N-连接聚糖的空间竞争而部分展开。两种小结合剂的多价形式在表达TLR3的细胞系中诱导NF-κB信号传导，表明它们可能具有治疗相关的生物学活性。我们的工作为开发特异性、稳定且易于制剂的基于蛋白质的TLR和其他模式识别受体激动剂奠定了基础。
