Spontaneous and specific chemical cross-linking in live cells to capture and identify protein interactions

Abstract

Covalently locking interacting proteins in situ is an attractive strategy for addressing the challenge of identifying weak and transient protein interactions, yet it is demanding to execute chemical reactions in live systems in a biocompatible, specific, and autonomous manner. Harnessing proximity-enabled reactivity of an unnatural amino acid incorporated in the bait toward a target residue of unknown proteins, here we genetically encode chemical cross-linkers (GECX) to cross-link interacting proteins spontaneously and selectively in live cells. Obviating an external trigger for reactivity and affording residue specificity, GECX enables the capture of low-affinity protein binding (affibody with Z protein), elusive enzyme-substrate interaction (ubiquitin-conjugating enzyme UBE2D3 with substrate PCNA), and endogenous proteins interacting with thioredoxin in E. coli cells, allowing for mass spectrometric identification of interacting proteins and crosslinking sites. This live cell chemistry-based approach should be valuable for investigating currently intangible protein interactions in vivo for better understanding of biology in physiological settings.

Proximity-enhanced SuFEx chemical cross-linker for specific and multitargeting cross-linking mass spectrometry

“…Recently, we developed proximity-enabled bioreactivity (13)(14)(15)(16), which allows unnatural amino acids (Uaas) bearing biocompatible functional groups to react with specific natural residues of proteins selectively by bringing the two residues into proximity (17). This methodology has enabled us to capture weak PPIs and transient enzyme-substrate interactions (12). In particular, a sulfonyl-fluoride-containing Uaa is able to react with Lys and His (18), and a fluorosulfatecontaining Uaa FSY reacts with Lys, His, and Tyr, both via sulfur-fluoride exchange (SuFEx) reactions (19).…”

“…An outstanding challenge for studying PPIs and their networks is to identify weak and transient protein interactions. We previously developed GECX, which uses a bioreactive Uaa to capture such interactions in situ for subsequent MS identification (12). However, as a single cross-linked peptide is generated for each interacting protein in GECX, which may or may not be identifiable by tandem MS, the number of identified proteins with direct cross-linked peptides remains low.…”

“…Using GECX, we genetically incorporated Uaa O-(3-bromopropyl)-L-tyrosine (BprY, Fig. 6A) into thioredoxin (Trx) to capture Trx-interacting proteins in E. coli cells (12,15). Whenever a substrate protein of Trx interacts with Trx, BprY reacts with the Cys residue of the substrate protein via proximity-enhanced reactivity, thus covalently cross-linking the substrate protein with Trx in vivo.…”

“…However, the possibility of excessive cross-linking dramatically complicates the data analysis, which remains a daunting challenge for CXMS, and high-density cross-linking may artificially distort protein tertiary structure as well (4,5). In addition, photo-cross-linkers generally have short half-lives and thus limited use in studying weak and transient protein-protein interactions (PPIs) (12). Clearly, new chemical cross-linking strategies that are Significance Chemical cross-linking mass spectrometry is a powerful method to identify protein interaction partners.…”

“…We further showed that NHSF could cross-link complex Escherichia coli whole-cell lysate revealing cross-linked peptides undetectable with existing reagents. Finally, we showed that this approach can enhance the identification of weak and transient protein interactions when combined with genetically encoded chemical cross-linking (GECX) (12).…”