Abstract
Proteins regulate biological functions through the formation of distinct protein complexes. Identification and characterization of these protein-protein interactions are critical to deciphering their mechanism of action. Different antibody-based or cross-linking-based methods have been developed to identify the protein-protein interactions. However, these methods require genetic engineering or other means to disrupt the native environments. To circumvent this limitation, we introduce here SMAP3-ID (small-molecule-assisted identification of protein-protein interactions through proximity) method to identify protein-protein interactions in native cellular environment. This method combines a selective ligand for binding to a protein of interest for photo-cross-linking, a live-cell-compatible bioorthogonal click reaction with a trifunctional chemical probe, and a final photo-cross-linking reaction to covalently capture the interacting proteins. Using the SMAP3-ID method and nuclear lamins as an example, we identified numerous lamin interactors in native cells. Significantly, we identified a number of mitochondrial enzymes as novel lamin A (LA) interactors. The interactions between mitochondrial enzymes and LA were further validated, which provides mechanistic insights underlying the metabolic alterations caused by mutations in LA. Furthermore, our previously described small-molecule ligand for LA, LBL1, also induced changes in mitochondrial activity and cellular bioenergetic organization. We conclude that SMAP3-ID is a potentially powerful and generalizable method to identify protein-protein interactions in the native cellular environment.