A nanobody-based approach to capture and visualize interactions of binary protein complexes in living cells

Protein function depends on interactions with other protein partners, ultimately leading to the formation of intricate protein-protein interaction (PPI) networks. These molecular networks (or interactomes) are formed progressively, each interaction influencing the next one. Accordingly, a same protein can lead to the formation of different interactomes depending on its first associated cofactor. Therefore, capturing PPIs of specific dimeric protein complexes is key for understanding the molecular rules underlying the diverse cell- and/or subcellular- functions of a bait protein of interest. Here, we introduce an innovative method called Bi-nano-ID that is based on bicolor bimolecular fluorescence complementation and the specific binding of a nanobody fused to a proximity-dependent biotinylating enzyme to tackle this issue. Bi-nano-ID was used to capture endogenous interactomes of the cytoplasmic TAZ/14-3-3e and nuclear TAZ/TEAD2 complexes, which are major downstream effectors of the Hippo signaling pathway. Among the different specific interactions, we revealed the role of a particular family of protease inhibitors for stabilizing and promoting the proliferative activity of TAZ/14-3-3e complexes in mesenchymal stem cells. Overall, our work establishes a novel sensitive method for capturing and visualizing specific interactions of binary bait protein complexes in human living cells.