Genetically Encoded Residue-Selective Photo-Crosslinker to Capture Protein-Protein Interactions in Living Cells

•Genetically encoded lysine-selective protein photo-crosslinker in living cells•Capturing protein interactions with lysine-selectivity in a temporal-resolved manner•Producing residue-predictable photo-crosslinking site and crosslinked peptides•Capture of elusive enzyme-substrate interaction and validation of lysine PTM site Protein-protein interactions play pivotal roles in virtually all cellular processes. The identification of transient and weak protein interactions in living cells still remains challenging with traditional strategies. Combining advantages of temporal control and the unique ability of residue selectivity, photo-crosslinkers offer are a powerful tool for identification of dynamic, weak, and transient protein interactions in living cells with specificity, accuracy, and reliability, thus, for better understanding of biological processes and pathophysiology. Investigating photo-crosslinking with residue selectivity may lead to the discovery of new photo-crosslinkers and help in expanding the repertoire of current protein crosslinking technology and interactive proteomics. Consequently, this approach will have an effect on a wide range of fields involving protein-protein and ligand-receptor interactions such as basic biological studies, interactive proteomics, protein structural studies, and chemical biology. The genetically encoded non-selective photo-crosslinkers suffer from unpredictable crosslinking sites and false positive interacting proteins, thus, the identification of protein interactions and mapping accurate interaction interfaces remain largely elusive in living systems. Here, we report genetically encoded, residue-selective protein photo-crosslinker enabling covalent crosslinking of the interacting proteins with proximal lysine, upon UV light activation in vitro and in living cells. Using this photo-crosslinker, we demonstrate a crosslinking strategy to capture protein-protein interactions with residue selectivity in a temporal-resolved manner. In addition, this crosslinking strategy produces the lysine-selective crosslinking site and predictable crosslinked peptide, which serve as the direct evidence for protein-protein interactions and facilitate mass spectrometry analysis. More importantly, this photo-crosslinker enables the capture of elusive enzyme-substrate interaction with directly interacting lysine and validation of acetylation site of the substrate. This strategy represents higher spatiotemporal resolution, accuracy, and reliability for investigating dynamic, transient, and weak protein-protein interactions in living systems. The genetically encoded non-selective photo-crosslinkers suffer from unpredictable crosslinking sites and false positive interacting proteins, thus, the identification of protein interactions and mapping accurate interaction interfaces remain largely elusive in living systems. Here, we report genetically encoded, residue-selective protein photo-crosslinker enabling covalent crosslinking of the interacting proteins with proximal lysine, upon UV light activation in vitro and in living cells. Using this photo-crosslinker, we demonstrate a crosslinking strategy to capture protein-protein interactions with residue selectivity in a temporal-resolved manner. In addition, this crosslinking strategy produces the lysine-selective crosslinking site and predictable crosslinked peptide, which serve as the direct evidence for protein-protein interactions and facilitate mass spectrometry analysis. 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To our delight, we found that o-NBAK reacted rapidly with lysine after 10 min of UV light activation, providing stable indazolone as ligation product with high yield (>70%, detected by HPLC), within 1 h incubation at 37°C, in buffer conditions (Figures S1 and S2). In contrast, no detectable stable ligation products were found from the reactions of aryl-nitroso compound and other natural amino acids with different sidechains (Table S1; Figure S3).45Lo Conte M.L. Carroll K.S. Chemoselective ligation of sulfinic acids with aryl-nitroso compounds.Angew. Chem. Int. Ed. 2012; 51: 6502-6505Crossref PubMed Scopus (53) Google Scholar The photogenerated aryl-nitroso intermediate could be detected on UPLC-MS analysis process (Figure S2) and half-life is more than 30 min in buffer conditions, which is more stable than other photogenerated reactive species from Uaas.14Nguyen T.A. Cigler M. Lang K. Expanding the genetic code to study protein-protein interactions.Angew. Chem. Int. 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In absence of o-NBAK, no full-length GST protein was detected (Figure 3B), when 1 mM o-NBAK was added in growth media, full-length GST was obtained in good yield (ca. 4.0 mg L−1, Figure S14). The purified GST-97o-NBAK was analyzed by electrospray ionization time-of-flight MS (ESI-TOF MS) (Figure 3C). The masses indicate that only o-NBAK was incorporated in GST and no peaks corresponding to other amino acids were observed. Furthermore, GST-97o-NBAK protein was digested with trypsin and analyzed by tandem MS (Figure 3D), a series of y and b ions clearly indicate that o-NBAK was incorporated at position 97, specified by the TAG codon. These results demonstrate that mutated MmPylRS (Y306A/Y384F) can efficiently recognize o-NBAK (Figure S14) and only o-NBAK was incorporated at TAG-encoded position in GST, and that o-NBAK was stable during protein synthesis in E. coli and protein purification process. As proof of principle, we chose to crosslink GST homodimer as o-NBAK enabling photo-crosslinking with proximal lysine inter-molecularly (Figure 4A). Based on GST dimer crystal structure, A97 located at a distance of ∼15 Å from a lysine residue (Lys44) from the opposite monomer, which indicated that o-NBAK at position 97 would be able to generate covalent linkage with Lys44 between GST dimer.29Yang B. Wu H. Schnier P.D. Liu Y. Liu J. Wang N. DeGrado W.F. Wang L. Proximity-enhanced SuFEx chemical cross-linker for specific and multitargeting cross-linking mass spectrometry.Proc. Natl. Acad. Sci. USA. 2018; 115: 11162-11167Crossref PubMed Scopus (42) Google Scholar, 30Tian Y. Jacinto M.P. Zeng Y. Yu Z. Qu J. Liu W.R. Lin Q. Genetically encoded 2-aryl-5-carboxytetrazoles for site-selective protein photo-