A Genetically Encoded aza-Michael Acceptor for Covalent Cross-Linking of Protein–Receptor Complexes

Abstract

Selective covalent bond formation at a protein–protein interface potentially can be achieved by genetically introducing into a protein an appropriately “tuned” electrophilic unnatural amino acid that reacts with a native nucleophilic residue in its cognate receptor upon complex formation. We have evolved orthogonal aminoacyl-tRNA synthetase/tRNA_CUA pairs that genetically encode three aza-Michael acceptor amino acids, <i>N</i>^ε-acryloyl-(<i>S</i>)-lysine (AcrK, <b>1</b>), <i>p</i>-acrylamido-(<i>S</i>)-phenylalanine (AcrF, <b>2</b>), and <i>p</i>-vinylsulfonamido-(<i>S</i>)-phenylalanine (VSF, <b>3</b>), in response to the amber stop codon in Escherichia coli. Using an αErbB2 Fab-ErbB2 antibody-receptor pair as an example, we demonstrate covalent bond formation between an αErbB2-VSF mutant and a specific surface lysine ε-amino group of ErbB2, leading to near quantitative cross-linking to either purified ErbB2 <i>in vitro</i> or to native cellular ErbB2 at physiological pH. This efficient biocompatible reaction may be useful for creating novel cell biological probes, diagnostics, or therapeutics that selectively and irreversibly bind a target protein <i>in vitro</i> or in living cells