Abstract
Advances in chemoproteomic technology have revealed covalent interactions between small molecules and protein nucleophiles, primarily cysteine, on a proteome-wide scale. Most chemoproteomic screening approaches are indirect, relying on competition between electrophilic fragments and a minimalist electrophilic probe with inherently limited proteome coverage. Here we develop a chemoproteomic platform for direct electrophile-site identification based on enantiomeric pairs of clickable arylsulfonyl fluoride probes. Using stereoselective site modification as a proxy for ligandability in intact cells, we identify 634 tyrosines and lysines within functionally diverse protein sites, liganded by structurally diverse probes. Among multiple validated sites, we discover a chiral probe that modifies Y228 in the MYC binding site of the epigenetic regulator WDR5, as revealed by a high-resolution crystal structure. A distinct chiral probe stimulates tumour cell phagocytosis by covalently modifying Y387 in the recently discovered immuno-oncology target APMAP. Our work provides a deep resource of ligandable tyrosines and lysines for the development of covalent chemical probes.
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Data availability
Uncropped gel images are provided as Source Data files accompanying this paper. The reported crystal structure has been deposited to the Protein Data Bank (PDB) under accession no. 8F93. All proteomic raw data have been deposited to MassIVE (http://massive.ucsd.edu) with accession no. MSV000090778, as well as in ProteomeXchange (http://www.proteomexchange.org) with accession no. PXD042307. Source data are provided with this paper.
Code availability
The script used for identifying modified sites and proximal ligands in the PDB has been deposited to GitHub (https://github.com/aacuesta/Ying2023NatChem).
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Acknowledgements
Funding for this study was provided by the Ono Pharma Foundation (J.T.), Pfizer and the Tobacco-Related Disease Research Program Postdoctoral Fellowship Award (T32FT4880 to G.B.C.). The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. We thank B. Küster for guidance on MS data acquisition and analysis.
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Authors and Affiliations
Contributions
Y.C. and J.T. conceived the project, designed the experiments and analysed the data. S.Z. and Y.S.M. synthesized the SFs. Y.C. performed the chemoproteomic and validation experiments. G.B.C. solved the WDR5 X-ray structure and performed biochemical experiments with recombinant APMAP. A.C. wrote the script for identifying modified sites in the PDB. R.A.K. performed the phagocytosis assays, with input from M.C.B. Y.C. and J.T. wrote the manuscript, with input from all of the authors.
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J.T. is a cofounder of Kezar Life Sciences and Terremoto Biosciences, and a scientific advisor to Entos. M.C.B. and R.A.K. have outside interests in DEM Biopharma. S.Z. and Y.S.M. are employees of Enamine Ltd and Chemspace LLC, respectively. The remaining authors declare no competing interests.
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Extended data
Extended Data Fig. 1 Volcano plots showing modified sites (MS2 and RTS-MS3 data) for probes 1–10.
a-j, Log2ratio((R)/(S)) is on the x-axis and –log10(P value) is on the y-axis. Modified sites are indicated as (R)-selective (blue), (S)-selective (orange), and nonselective (grey). P values were determined by Student’s t-test (two-tailed, two-sample equal variance).
Extended Data Fig. 2 Analysis of tyrosines and lysines modified by chiral sulfonyl fluoride probes.
a-f, Number of liganded and total Tyr and Lys identified in this study compared with the indicated previous studies14,15. g, The number of enantiomeric probe pairs (based on fragments 1-10) per site (x- axis) was plotted against the total number of sites modified by the indicated number of enantiomeric probe pairs (y-axis).
Extended Data Fig. 3 Functionally diverse protein sites targeted by chiral sulfonyl fluoride probes.
a-f, Structures of FUS, YBX1, KIF11, KDM1A, PRMT5, and SIRT2, highlighting the probe-modified Tyr proximal to bound ligands.
Extended Data Fig. 4 Probe modification of LCMT1.
a, HEK293T cells were transfected with Flag-LCMT1 and treated with SF probes (10 μM, 1 h). After cell lysis, anti-Flag enrichment, 3xFlag peptide elution and click reaction with TAMRA-azide, samples were analyzed by in-gel fluorescence and western blotting. NC indicates nontransfected cells. Data are representative of two independent experiments. b, HEK293T cells were transfected with WT or Y315F Flag- LCMT1 and treated with 4-SF probes (10 μM, 1 h). The cells were lysed and processed as described above. Data are representative of two independent experiments.
Extended Data Fig. 5 Probe modification of ABR.
a, HEK293T cells were transfected with Flag-ABR and treated with SF probes (10 μM, 1 h). After cell lysis, anti-Flag enrichment, 3xFlag peptide elution and click reaction with TAMRA-azide, samples were analyzed by in-gel fluorescence and western blotting. NC indicates nontransfected cells. Data are representative of two independent experiments. b, HEK293T cells were transfected with WT or Y344F Flag-ABR and treated with 7-SF probes (10 μM, 1 h). The cells were lysed and processed as described above. Data are representative of two independent experiments.
Extended Data Fig. 6 Probe modification of WDR82.
a, HEK293T cells were transfected with WT or Y102F Flag-WDR82 and treated with 7-SF probes (50 μM, 1 h). After cell lysis, anti- Flag enrichment, 3xFlag peptide elution and click reaction with TAMRA-azide, samples were analyzed by in-gel fluorescence and western blotting. Data are representative of two independent experiments. NC indicates nontransfected cells. b, Recombinant WT and Y102F WDR82 (0.44 μM) were incubated with 7-SF probes (40 μM, 37 °C, overnight) and analyzed by intact-protein mass spectrometry. Data are representative of two independent experiments. c, HEK293T cells were transiently transfected with Flag-WDR82 and treated with the indicated SF probes (50 μM, 1 h). The cells were lysed and processed as described above. Data are representative of two independent experiments. d, Overlay of the AlphaFold structure of WDR82 with the crystal structure of WDR5 bound to KANSL1 peptide (PDB: 4CY2).
Extended Data Fig. 7 Electron density map of (R)-2-SF.
Electron density (2Fo-Fc) map of (R)-2-SF is shown at a contour level of 1σ.
Extended Data Fig. 8 Data collection and refinement statistics.
Data collection and refinement statistics (molecular replacement) for WDR5-(R)-2-SF complex.
Extended Data Fig. 9 Chemoproteomic analysis of 1-SF probes in dose-response mode.
a, HEK293T cells that stably overexpress Flag-APMAP were treated separately with 0, 3.75, 7.5, 15, 30 and 60 µM of (R)-1-SF and (S)-1-SF for 1.5 h at 37 °C. Cell lysates were conjugated with biotin–DADPS-azide via click chemistry. After enrichment and trypsin digestion, probe-modified peptides from each sample were separately labeled with TMT 11-plex reagents. Probe-modified peptides from each sample were eluted with 2% formic acid, combined, and analyzed by LC-MS/MS. b-d, TMT reporter ion intensities were plotted as a function of probe concentration for peptides quantified by MS2 and RTS: APMAP Y387 (b), APMAP K347 (c), and MTHFD1 Y52 (d). e, Crystal structure (PDB: 1DIA) depicting MTHFD1 Y52 proximal to the inhibitor LY249543.
Extended Data Fig. 10 Antibody-dependent phagocytosis assay.
a, APMAPKO Ramos- Cas9 cells (rescued with Flag-APMAP WT, E103A and Y387F) were resuspended in SDS loading buffer containing 100 mM DTT, sonicated and heated (70 °C, 10 min). Samples were analyzed by western blotting. Data are representative of two independent experiments. b, Scheme of phagocytosis assay.
Supplementary information
Supplementary Information
Supplementary dataset legends, tables, biological methods, synthetic methods, NMR spectra, references.
Supplementary Data 1
MS data from TMT6 experiments. (Table 1) Modified sites and enantiomer modification ratios for 10 chiral sulfonyl fluoride probe pairs. (Tables 2–21) Each table displays the MS data, including TMT6 intensity values (Maxquant output), for sites modified by the indicated chiral sulfonyl fluoride probe pair (MS2 and RTS methods). P-values were determined by Student’s t-test (two-tailed, two-sample equal variance).
Supplementary Data 2
MS data from the 1-SF dose-response experiment (Maxquant output), related to Extended Data Fig. 9. (Table 1) MS2 method. (Table 2) RTS method.
Supplementary Data 3
Sequences of oligonucleotides used in this work.
Source data
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Source Data Extended Data Fig. 4
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Source Data Extended Data Fig. 10
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Chen, Y., Craven, G.B., Kamber, R.A. et al. Direct mapping of ligandable tyrosines and lysines in cells with chiral sulfonyl fluoride probes. Nat. Chem. 15, 1616–1625 (2023). https://doi.org/10.1038/s41557-023-01281-3
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DOI: https://doi.org/10.1038/s41557-023-01281-3
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