Engineering Nanobody Conformational Switches

Conformational signaling is widely used in biology, but remains difficult to engineer into proteins. Here, we explore ligand-induced folding of nanobodies as a general strategy to generate conformational receptors for a broad range of target ligands. More specifically, we have generated partially unfolded nanobody variants that fold exclusively upon binding to their target ligand. To do so, we have introduced destabilizing mutations in the framework region, carefully tuning their stability while preserving molecular recognition. Given the high conservation of the framework across different nanobodies, this approach is broadly applicable to many other target ligands, which has enabled us to engineer conformational switches for three different important biomarkers and drugs: the pregnancy hormone chorionic gonadotropin, the cytokine tumor necrosis factor alpha, and the chemotherapy and immunosuppressant drug methotrexate. Another useful property of conformational signaling is that it can directly gate signal transduction, which we have leveraged to build an optical sensor for the quantitative detection of the pregnancy hormone in urine. Finally, conformational signaling introduces new mechanisms for the allosteric modulation of binding, which we have explored to control the affinity and dynamic range of the sensor. Our work thus establishes a rational strategy for engineering conformational signaling receptors with customizable properties and generalizable for many different target ligands.