Affinity-guided covalent conjugation reactions based on PDZ-peptide and SH3-peptide interactions

Specific protein-peptide interactions are prevalent in the living cells and form a tightly regulated signaling network. These interactions, many of which have structural information revealed, provide ideal templates for affinity-guided covalent bioconjugation. Here we report the development of a set of four new reactions that covalently and site-specifically link nonenzymatic scaffolding domains (two PDZ and two SH3 domains) and their ligands through thiol-chloroacetyl SN2 reaction. Guided by the three-dimensional structure of the wild type complex, a selected position of the protein was mutated to cysteine, and at the same time, an α-chloroacetyl group was installed at a corresponding position of the peptide. Specific binding interaction between the two brings the reactive groups into close proximity, converts the nonreactive cysteine residue into a content-dependent reactive site, and induces the nucleophilic reaction that is inert in the absence of the binding event. The specificity, orthogonality, and modularity of the four reactions were characterized, the reaction was applied to label proteins in vitro and receptor on the surface of mammalian cells, and the system was utilized to assemble covalent protein complexes with unnatural geometries.