A Genetically Encoded Fluorosulfonyloxybenzoyl-l-lysine for Expansive Covalent Bonding of Proteins via SuFEx Chemistry

Abstract

Genetically introducing novel chemical bonds into proteins provides innovative avenues for biochemical research, protein engineering, and biotherapeutic applications. Recently, latent bioreactive unnatural amino acids (Uaas) have been incorporated into proteins to covalently target natural residues through proximity-enabled reactivity. Aryl fluorosulfate is particularly attractive due to its exceptional biocompatibility and multitargeting capability via sulfur­(VI) fluoride exchange (SuFEx) reaction. Thus far, fluorosulfate-l-tyrosine (FSY) is the only aryl fluorosulfate-containing Uaa that has been genetically encoded. FSY has a relatively rigid and short side chain, which restricts the diversity of proteins targetable and the scope of applications. Here we designed and genetically encoded a new latent bioreactive Uaa, fluorosulfonyloxybenzoyl-l-lysine (FSK), in E. coli and mammalian cells. Due to its long and flexible aryl fluorosulfate-containing side chain, FSK was particularly useful in covalently linking protein sites that are unreachable with FSY, both intra- and intermolecularly, in vitro and in live cells. In addition, we created covalent nanobodies that irreversibly bound to epidermal growth factor receptors (EGFR) on cells, with FSK and FSY targeting distinct positions on EGFR to counter potential mutational resistance. Moreover, we established the use of FSK and FSY for genetically encoded chemical cross-linking to capture elusive enzyme–substrate interactions in live cells, allowing us to target residues aside from Cys and to cross-link at the binding periphery. FSK complements FSY to expand target diversity and versatility. Together, they provide a powerful, genetically encoded, latent bioreactive SuFEx system for creating covalent bonds in diverse proteins in vitro and in vivo, which will be widely useful for biological research and applications.

Accelerating PERx Reaction Enables Covalent Nanobodies for Potent Neutralization of SARS-Cov-2 and Variants

“…To react with residues at further distances, we have also developed FSK that has a longer and more flexible side chain with similar reactivity. 44 These site identification strategies are straightforward and obviate sophisticated instrumentation such as mass spectrometry, and should be generally applicable to engineering various proteins such as antibodies, Fab, single-chain variable fragment (ScFv), affibodies, DARPins and so on, into covalent binders.…”

“…Our results demonstrate that nanobodies can be readily engineered into covalent binders through incorporating a latent bioreactive Uaa, which reacts with a natural residue of the target protein only upon nanobody-target binding, expanding the scope of PERx that we previously developed and applied to the immune-checkpoint PD-1/PD-L1. 19 20 Sites in nanobody appropriate for FSY or FFY incorporation and cross-linking were identified either by inspecting the structure of nanobody-target complex 44 or simply screening all sites in nanobody’s three CDRs. Recent breakthrough in accurate prediction of protein structure and interactions have made structural information available for a broad range of proteins, 45 46 which will greatly facilitate structure-guided site identification.…”

Spontaneous Orthogonal Protein Crosslinking via a Genetically Encoded 2‐Carboxy‐4‐Aryl‐1,2,3‐Triazole

“…Our design involves the introduction of a genetically encoded electrophilic amino acid site‐specifically into a protein of interest, which then undergoes spontaneous, intramolecular, proximity‐driven crosslinking with a nearby nucleophilic residue. While several electrophilic amino acids have been incorporated into proteins site‐specifically through genetic code expansion, [3] including p ‐2′‐fluoroacetyl‐phenylalanine, [4] bromoalkyl amino acids BprY [5] and BrC6K, [6] fluorosulfate‐modified tyrosine (FSY) [7] and lysine (FSK), [8] and noncanonical amino acids containing perfluorobenzene [9] and vinyl sulfonamide, [10] they preferentially react with cysteine and lack orthogonality to the disulfide bond. Recently, Schultz and co‐workers reported site‐specific incorporation of 4‐fluorophenylcarbamate‐lysine (FPheK) into thioredoxin [11] .…”

A chemical tool for blue light-inducible proximity photo-crosslinking in live cells

“…1a). For interactome mapping, genetically encoded chemical crosslinkers have also been used to capture PPI networks and simultaneously capture the nucleophilic residues of nearby proteins for crosslinking [8][9][10] although this system requires the expression of multiple subunits for the metabolic incorporation of unnatural amino acid (UAA).…”