Uncover New Reactivity of Genetically Encoded Alkyl Bromide Non-Canonical Amino Acids

Abstract

Genetically encoded non-canonical amino acids (ncAAs) with electrophilic moieties are excellent tools to investigate protein-protein interactions (PPIs) both in vitro and in vivo. These ncAAs, including a series of alkyl bromide-based ncAAs, mainly target cysteine residues to form protein-protein cross-links. Although some reactivities towards lysine and tyrosine residues have been reported, a comprehensive understanding of their reactivity towards a broad range of nucleophilic amino acids is lacking. Here we used a recently developed OpenUaa search engine to perform an in-depth analysis of mass spec data generated for Thioredoxin and its direct binding proteins cross-linked with an alkyl bromide-based ncAA, BprY. The analysis showed that, besides cysteine residues, BprY also targeted a broad range of nucleophilic amino acids. We validated this broad reactivity of BprY with Affibody/Z protein complex. We then successfully applied BprY to map a binding interface between SUMO2 and SUMO-interacting motifs (SIMs). BprY was further applied to probe SUMO2 interaction partners. We identified 264 SUMO2 binders, including several validated SUMO2 binders and many new binders. Our data demonstrated that BprY can be effectively used to probe protein-protein interaction interfaces even without cysteine residues, which will greatly expand the power of BprY in studying PPIs.

Yeast Display Enables Identification of Covalent Single-Domain Antibodies against Botulinum Neurotoxin Light Chain A

“…A similar analysis involving the nearest LC/A nucleophile revealed that crosslinking only occurred with Cα–Cα distances between 8–10 Å, although not all mutants with a nucleophilic residue at this distance showed evidence of spontaneous crosslinking (Figure S8b). Since alkyl halides have been shown to react with diverse nucleophiles present in proteins, , we also systematically examined the Cα distances to each type of reactive nucleophile in LC/A (Figure S8c). No discernible correlation was observed between crosslinking and distance for most nucleophiles.…”

Mapping protein direct interactome of oxidoreductases with small molecular chemical cross-linkers in live cells

“…E) KEGG analysis of Trx1 interacting proteins from this study. Comparison of identified cross-linked peptides and interacting proteins from this study with those identified using an unnatural amino acid BprY (F) [ 29 ], disulfide trapping(G) [ 5 ], and SNO (H) [ 30 ]. …”

“…Previously, we identified 121 cross-linked peptides by incorporating genetically encoded unnatural amino acid BprY onto residue 33 of Trx1 [ 29 ], leading to the discovery of 87 interacting proteins. The study here covered ∼32% BprY cross-linked peptides and ∼48% BprY cross-linked proteins ( Fig.…”

Genetically encoded crosslinkers to address protein–protein interactions

“…Among those, bromoalkane‐ncAAs have been reported to also target, besides the preferred Cys residue, the primary amine of Lys, the imidazole of His, and the acid group of Glu and Asp. A recent comprehensive analysis of the reactivity of BrPrY ( 11a ) based on the mass spectrometric characterization of crosslinking products has added to the list the alcohol group of Ser, Thr, Tyr (Shu et al, 2022 ). An interesting ncAA targeting Cys is ThioD ( 14 ), which carries on the side chain a thioester suitable for chemical ligation (Xuan et al, 2018 ).…”