Fig. 1.

Spontaneous intermolecular amide bond formation by SpyTag. (A) Amide bond formation between Lys and Asp side chains. (B) Key residues for amide bond formation in CnaB2 shown in stick format, based on PDB 2X5P. (C) Cartoon of SpyTag construction. Streptococcus pyogenes (Spy) CnaB2 was dissected into a large N-terminal fragment (SpyCatcher, left) and a small C-terminal fragment (SpyTag, right). Reactive residues are highlighted in red. (D) SpyTag and SpyCatcher associated covalently. SpyTag-MBP and SpyCatcher were mixed each at 10 μM for 3 h and analyzed after boiling by SDS-PAGE with Coomassie staining, alongside unreactive controls, SpyCatcher E77Q (EQ) and D117A (DA) SpyTag-MBP.