Abstract
Genetic code expansion enables site-specific photo-crosslinking by introducing photo-reactive non-canonical amino acids into proteins at defined positions during translation. This technology is widely used for analyzing protein-protein interactions and is applicable in mammalian cells. However, the identification of the crosslinked region still remains challenging. Here, we developed a new method to identify the crosslinked region by pre-installing a site-specific cleavage site, an α-hydroxy acid (Nε -allyloxycarbonyl-α-hydroxyl-l-lysine acid, AllocLys-OH), into the target protein. Alkaline treatment cleaves the crosslinked complex at the position of the α-hydroxy acid residue and thus helps to identify which side of the cleavage site, either closer to the N-terminus or C-terminus, the crosslinked site is located within the target protein. A series of AllocLys-OH introductions narrows down the crosslinked region. By applying this method, we identified the crosslinked regions in lysosomal-associated membrane protein type 2A (LAMP2A), a receptor of chaperone-mediated autophagy, in mammalian cells. The results suggested that at least two interfaces are involved in the homophilic interaction, which requires a trimeric or higher oligomeric assembly of adjacent LAMP2A molecules. Thus, the combination of site-specific crosslinking and site-specific cleavage promises to be useful for revealing binding interfaces and protein complex geometries.