Site‐specific photo‐crosslinking/cleavage for protein–protein interface identification reveals oligomeric assembly of lysosomal‐associated membrane protein type 2A in mammalian cells

Abstract

AbstractGenetic 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.