Proximity-Dependent Labeling of Cysteines

Mapping protein–protein interactions is crucial for understanding various signaling pathways in living cells, and developing new techniques for this purpose has attracted significant interest. Classic methods (e.g., the yeast two-hybrid) have been supplanted by more sophisticated chemical approaches that label proximal proteins (e.g., BioID, APEX). Herein we describe a proximity-based approach that uniquely labels cysteines. Our approach exploits the nicotinamide N-methyltransferase (NNMT)-catalyzed methylation of an alkyne-substituted 4-chloropyridine (SS6). Upon methylation of the pyridinium nitrogen, this latent electrophile diffuses out of the active site and labels proximal proteins on short time scales (≤5 min). We validated this approach by identifying known (and novel) interacting partners of protein arginine deiminase 2 (PAD2) and pyruvate dehydrogenase kinase 1 (PDK1). To our knowledge, this technology uniquely exploits a suicide substrate to label proximal cysteines in live cells.