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.