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Single-electron transfer between sulfonium and tryptophan enables site-selective photo crosslinking of methyllysine reader proteins

Nature Chemistry volume 16, pages 1267–1277 (2024)Cite this article

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Abstract

The identification of readers, an important class of proteins that recognize modified residues at specific sites, is essential to uncover the biological roles of post-translational modifications. Photoreactive crosslinkers are powerful tools for investigating readers. However, existing methods usually employ synthetically challenging photoreactive warheads, and their high-energy intermediates generated upon irradiation, such as nitrene and carbene, may cause substantial non-specific crosslinking. Here we report dimethylsulfonium as a methyllysine mimic that binds to specific readers and subsequently crosslinks to a conserved tryptophan inside the binding pocket through single-electron transfer under ultraviolet irradiation. The crosslinking relies on a protein-templated σ–π electron donor–acceptor interaction between sulfonium and indole, ensuring excellent site selectivity for tryptophan in the active site and orthogonality to other methyllysine readers. This method could escalate the discovery of methyllysine readers from complex cell samples. Furthermore, this photo crosslinking strategy could be extended to develop other types of microenvironment-dependent conjugations to site-specific tryptophan.

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Fig. 1: Overview of strategies of crosslinking to methyllysine readers.
Fig. 2: H3K9NleS+me2 peptide binds to CBX1 and selectively crosslinks to the tryptophan in the binding pocket.
Fig. 3: Mechanistic study of photo crosslinking between H3K9NleS+me2 peptide and CBX1.
Fig. 4: NleS+me2 peptide probes crosslink to methyllysine-binding proteins broadly with high specificity.
Fig. 5: Investigation of crosslinked proteins by NleS+me2 peptide probe in cell nuclei.
Fig. 6: Scope expansion of photo-induced crosslinking reaction between sulfonium and tryptophan.

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Data availability

Data that support the findings of this study are available in the Article and Supplementary Information. Raw proteomics data are deposited in the PRoteomics IDEntifications (PRIDE) database with accession numbers PXD051693 and PXD049149. Source data are provided with this paper.

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Acknowledgements

We acknowledge the support from the National Natural Science Foundation of China (no. 22161132006 to M.W.), Key R&D Program of Zhejiang (2024SSYS0036 to M.W.), Westlake University startup (to M.W.), National Natural Science Foundation of China (22322411 to L.Z.), National Key R&D Program of China (2021YFA1301501 to L.Z.) and Strategic Priority Research Program of the Chinese Academy of Sciences (XDB37040105 to L.Z.). We thank the Instrumentation and Service Center for Molecular Sciences (ISCMS) for the instrument support. In addition, we thank Y. Chen of ISCMS for the data acquisition and analysis of sulfonium compounds by mass spectrometry and Z. Chen of ISCMS for the characterization of UV light sources. We also thank the Biomedical Research Core Facilities including the Mass Spectrometry & Metabolomics Core Facility, High-throughput Core Facility and Protein Characterization and Crystallography Facility for data acquisition and analysis. We thank the Instrumentation and Service Center for Physical Sciences for supporting the ITC measurement. We thank Y. Wang at Westlake University for the helpful discussion of the crosslinking reaction mechanism. We thank S. Ma for the assistance with recombinant BPTF preparation.

Author information

Author notes

  1. These authors contributed equally: Feng Feng, Yingxiao Gao, Qun Zhao.

Authors and Affiliations

  1. Department of Chemistry, Zhejiang University, Hangzhou, China

    Feng Feng

  2. Department of Chemistry, School of Science, Westlake University, Hangzhou, China

    Feng Feng, Ting Luo, Qingyun Yang, Yihang Xiao, Yusong Han & Mingxuan Wu

  3. Department of Chemistry, Fudan University, Shanghai, China

    Yingxiao Gao

  4. State Key Laboratory of Medical Proteomics, National Chromatographic R. & A. Center, CAS Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, China

    Qun Zhao, Nan Zhao & Lihua Zhang

  5. Institute of Natural Sciences, Westlake Institute for Advanced Study, Hangzhou, China

    Qingyun Yang, Yihang Xiao & Mingxuan Wu

  6. Key Laboratory of Structural Biology of Zhejiang Province, School of Life Sciences, Westlake University, Hangzhou, China

    Jinheng Pan & Shan Feng

  7. Mass Spectrometry & Metabolomics Core Facility, The Biomedical Research Core Facility, Westlake University, Hangzhou, China

    Jinheng Pan & Shan Feng

  8. Westlake Laboratory of Life Sciences and Biomedicine, Hangzhou, China

    Mingxuan Wu

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Contributions

F.F. synthesized and characterized the small molecules and peptides. Y.G. and F.F. prepared the recombinant reader proteins and conducted crosslinking analysis. Y.G. carried out the reader-binding assays and crosslinking kinetic analysis. Q.Z., N.Z. and L.Z. designed and performed the crosslinking mass spectrometry. T.L. prepared the recombinant betaine- and choline-binding proteins. T.L. and Q.Y. performed the cell-based experiments. Q.Y. studied the crosslinking of antibodies. Y.X. conducted the chemical crosslinker assay of BRWD3. Y.X. and Y.H. designed and performed the NanoBiT crosslinking experiment. J.P. and S.F. conducted the top-down mass spectrometry analysis of CBX1 conjugate. M.W. designed and directed the work. M.W. wrote the manuscript with contributions from all authors. All authors prepared the figures, Methods and Supplementary Information and commented on the paper.

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Correspondence to Mingxuan Wu.

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Extended data

Extended Data Fig. 1 Measurement of binding affinity between recombinant readers in this study and FITC labeled methyllysine peptides by fluorescence polarization.

a, CBX1 and FITC-H3K9me3 peptide. b, MPP8 and FITC-H3K9me3 peptide. c, BPTF and FITC-H3K4me3 peptide. d, JMJD2A and FITC-H3K4me3 peptide. e, JMJD2A and FITC-H4K20me3 peptide. f, mORC1 and FITC-H4K20me2 peptide. Average values and errors ( ± s.e.m.) were calculated from n = 3 technical replicates.

Source data

Extended Data Fig. 2 Spectra of UV light source and sample absorption.

a, Emission spectrum from the UV-B lamp with 305 nm long pass filter in this study. b, Absorption spectra of CBX1, H3K9NleS+me2 peptide (5), and a mixture of CBX1 and peptide (5) in 100 mM HEPES (pH=7.5).

Source data

Extended Data Fig. 3 Additional data of binding and crosslinking activity of H3K9NleS+me2 peptide to CBX1.

a, Binding kinetics of interaction between CBX1 and H3K9NleS+me2 peptide (S16) by bio-layer interferometry, and steady-state graph is shown on the right. b, Mass spectrometry analysis of the crosslinking between CBX1 and H3K9NleS+me2 peptide (5) at time points. c,d, Mass spectrometry analysis of the crosslinking between CBX1 and peptide (5) by addition of TEMPO (7 mM) for 5 min or 30 min. e, Top-down mass spectrometry analysis of the methyl-CBX1 conjugate.

Source data

Extended Data Fig. 4 Characterization of binding and crosslinking activity of H3K9NleS+me2 peptide to MPP8.

a, 3D structure of H3K9me3 peptide and MPP8 (PDB: 3R93). b, High resolution mass spectrum of the reaction mixture of H3K9NleS+me2 peptide (5) and MPP8 under 20 min UV-B irradiation. c, Binding kinetics of interaction between MPP8 and H3K9NleS+me2 peptide (S16) by bio-layer interferometry.

Source data

Extended Data Fig. 5 Analysis of the reactivity between H3K9NleS+me2 peptide (5) and peptide or proteins without binding pocket of H3K9me3.

a, HPLC analysis of reaction mixture of H3K9NleS+me2 peptide (5) and a Tryptophan-containing short peptide (S3) under the standard crosslinking condition. Integration of the peptide (S3) peak did not change, and no crosslinked peptide product was observed. b-e, Mass spectrometry analysis of reaction mixture H3K9NleS+me2 peptide (5) and tryptophan-containing proteins under the standard crosslinking condition. Tryptophan residues are shown as stick in green.

Source data

Extended Data Fig. 6 Comparison of crosslinking activities by NleS+me2 peptide, NvaS+me2 peptide, and Met+me peptide.

The reader protein CBX1, BPTF and dSfmbt were applied to crosslinking by the sulfonium peptide with distinct side chain. The product yields were calculated based on the peak integrations from mass spectra as shown in Fig. 4e.

Source data

Extended Data Fig. 7 Investigation of crosslinked proteins by NleS+me2 peptide probe in cell nuclei.

a, Volcano plots of the crosslinked proteins from H3K9NleS+me2 probes (S16) with different competition by unmodified or Kme3 peptides. The hits in the plot with fold change>2 and p value < 0.05 are shown as red dots. Reported readers are highlighted by the name. P-values were determined by student’s t-test (two-tailed, two-sample equal variance). b, Characterization of crosslinking activities of BRWD3 W1100A mutant by H3K4NleS+me2 peptide (S14). Western blot experiment of independent replicates was repeated twice. c, Characterization of binding activities of W1100A mutant by chemical crosslinker. The assay was repeated twice with similar results. d, Predicted 3D structure of BRWD3 structure by AlphaFold. W1063 and W1089 are likely to bind methyllysine.

Source data

Extended Data Fig. 8 Detailed workflow of crosslinking mass spectrometry (XL-MS).

HeLa cell nuclei were extracted for crosslinking with sulfonium peptide probe under UV irradiation. The washed nuclei were lysed by sonication and the supernatant was applied for enrichment by streptavidin resin. The crosslinked proteins on resin were digested by GluC followed by trypsin. The released crosslinked peptide fragments were loaded to LC-MS/MS for data analysis and searching to identify crosslinked proteins and the specific tryptophan.

Extended Data Fig. 9 Additional data of sulfonium-mediated crosslinking to betaine and choline binding proteins.

a, 3D structure of betaine in OpuAC binding pocket. b, OpuAC, ProX, and ChoX were selectively crosslinked by the corresponding sulfonium analogues. Acetylcholine analogue and SAM were not active.

Source data

Extended Data Fig. 10 Photo-induced crosslinking reaction between LgBiT and sulfonium-SmBiT.

a, Structural analysis of NanoBiT. All aromatic residues were highlighted in pink. It demonstrated that W11 is not in an aromatic cage. b, Interface between LgBiT and SmBiT indicates that W11 is on LgBiT surface rather than inside a pocket. c, Mass spectrometry analysis of LgBiT and sulfonium myc-SmBiT (S25) crosslinking.

Source data

Supplementary information

Supplementary Information

Additional experimental procedures, NMR and mass spectra and so on.

Reporting Summary

Supplementary Data 1

Source data for crosslinking mass spectra.

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Source Data Fig. 4

Statistical source data and unprocessed western blots.

Source Data Fig. 5

Statistical source data and unprocessed western blots.

Source Data Fig. 6

Statistical source data and unprocessed gels and western blots.

Source Data Extended Data Fig. 1

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Source Data Extended Data Fig. 7

Statistical source data and unprocessed western blots.

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Feng, F., Gao, Y., Zhao, Q. et al. Single-electron transfer between sulfonium and tryptophan enables site-selective photo crosslinking of methyllysine reader proteins. Nat. Chem. 16, 1267–1277 (2024). https://doi.org/10.1038/s41557-024-01577-y

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