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/tRNACUA pairs that genetically encode three aza-Michael acceptor amino acids, Nε-acryloyl-(S)-lysine (AcrK, 1), p-acrylamido-(S)-phenylalanine (AcrF, 2), and p-vinylsulfonamido-(S)-phenylalanine (VSF, 3), 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 in vitro 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 in vitro or in living cells.

Enhancing protein stability with extended disulfide bonds

“…These longer and more flexible linkages should make it much easier to cross-link sites within proteins. Indeed, it has recently been shown that electrophilic amino acids with extended structures can selectively stabilize proteins and protein complexes by intra-and intermolecular cross-links, respectively (13,(23)(24)(25). However, the use of electrophilic NCAAs to form noncanonical cross-links in a bacterial selection system can be complicated by cellular toxicity, irreversible reactions, reaction rates, and scavenging by intracellular nucleophiles.…”

Bioconjugation of therapeutic proteins and enzymes using the expanded set of genetically encoded amino acids

“…Proximity-enabled reactivity of ''tuned'' electrophilic NNAAs towards nucleophilic natural amino acids can also be used to probe protein-protein interaction. Selective formation of irreversible covalent bond between a specific lysine of a cancer biomarker ErbB2 and VSF genetically incorporated into anti-ErbB2 Fab was confirmed in vitro and in vivo (Furman et al, 2014). This type of NNAAs has a promise for identifying unknown interacting partners of a target protein, especially those with a large dissociation constant.…”

“…As a result of UV irradiation, a covalently-linked GST dimer was formed when BZF was placed in the dimer interface, but no crosslinking occurred when placed outside the interface. Functional groups targeting specific natural amino acids in a spontaneous and proximity-enhanced manner have been genetically encoded through evolved pairs of amber suppressor tRNA/aaRS (VSF, BCK and ACK; Figure 2) Furman et al, 2014;Xiang et al, 2013;Xiang et al, 2014). These NNAAs have electrophilic residues that selectively react with proximal nucleophiles such as lysine, histidine and cysteine ( Figure 1).…”

Exploring the potential impact of an expanded genetic code on protein function

“…Unless otherwise mentioned, all other chemicals were purchased from Sigma-Aldrich and used without further purification. AcrF was synthesized as described (47). Absorbance spectra were measured with a Hewlett-Packard 8453 UV-visible spectrophotometer.…”