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

doi:10.21203/rs.3.rs-3149584/v1

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

https://doi.org/10.21203/rs.3.rs-3149584/v1

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published 30 Jul, 2024

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The identification of readers, an important class of proteins that recognize modified residues at specific sites, is essential to uncover biological roles of posttranslational 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 significant 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.

Physical sciences/Chemistry/Chemical biology/Chemical modification

Physical sciences/Chemistry/Chemical biology/Proteins

Physical sciences/Chemistry/Chemical biology/Post-translational modifications/Methylation

Posttranslational modifications (PTMs), including phosphorylation, acetylation, and methylation, play significant roles in regulation of protein functions¹. Reader proteins that recognize site-specific modified residues are an important class of proteins that control many cellular processes. For example, bromodomain that binds histone acetyllysine and YEATS domain that binds histone crotonyllysine promote open chromatin for active transcription^2–5. Therefore, investigation of readers is essential to understand mechanisms of PTMs.

Histone lysine methylation has critical functions of chromatin regulation, such as H3K9 methylation at heterochromatin for gene repression and H3K4me1 at enhancer for gene transcription⁶. Because lysine methylation does not alter the charge status and only slightly increases the size of lysine, this modification is believed to regulate protein function mainly by methyllysine readers⁷. Several domain classes have been identified and they share the same binding strategy to methyllysine by tryptophan-containing aromatic cages (Fig. 1a), including chromodomain, PHD domain, Tudor domain, BAH domain, and MBT domain, etc⁸. Because dysregulation of lysine methylation may drive cancer, many inhibitors of methyllysine reader proteins are under development for therapeutic purposes. However, some hypothetical readers of histone methyllysine have not been reported yet, including H3K56me3, H3K79me3, and H4K16me3, etc⁹. In addition, there is a large growing number of nonhistone methylation from state-of-the-art proteomic study, and therefore, there is a substantial gap between the number of methyllysine sites and the number of identified readers^10,11. As a result, investigation of novel methyllysine readers is urgent to comprehensively understand lysine methylation in cells.

In traditional biochemical experiments, methyllysine readers were identified by methyllysine peptides via pull down^12,13. Due to the binding affinity-based strategy, it is difficult to enrich readers with moderate/weak affinity or low abundance from cell samples. Alternatively, aryl azide or diazirine group is introduced to methyllysine peptide for photo crosslinking to potential proteins of interests via covalent bond (Fig. 1b)¹⁴. A recent study of H3K79me2 reader is a classic example, but this method has limitations¹⁵. To crosslink to readers, the photoreactive group should be proximate to methyllysine, but an additional group may interfere with the original binding between methyllysine and readers. As a result, the placement of photoreactive group on the probe requires laborious chemical synthesis of a series of probes for optimization¹⁶. In addition, the highly reactive intermediates such as nitrene and carbene from ultraviolet (UV) irradiation may cause significant non-specific crosslinking. As a result, a convenient synthetic probe that directly conjugates to the binding pocket with high site-selectivity would be desired to explore methyllysine readers.

Chemoselective conjugation to native proteins offers chemical diversity of protein tools. Plenty of amino acid selective reactions have been developed for distinct residues including tyrosine^17,18, tryptophan^19–24, etc, but the cases of site-selective conjugation depending on chemical microenvironment are limited (Fig. 1b). It is more challenging to target residues exclusively inside pockets due to low accessibility. Sulfonium is widely used by organic chemists in synthesis due to the inherent electron deficiency²⁵. Cells employ sulfonium in the form of S-Adenosyl methionine (SAM) as a cofactor for distinct types of biochemical reactions. SAM acts as a methyl group donor for methylation of proteins, DNAs and RNAs by methyltransferases via S_N2 substitution^26–28. SAM also acts as radical initiator by Fe-S cluster via reductive cleavage, and the resulting 5′-deoxyadenosyl radical plays significant roles in biosynthesis of natural product such as biotin and cyclic peptides^29,30. On account of the unique reactivity and compatibility under physiological condition, we envision that the positively charged sulfonium could bind to aromatic cages and react with electron-rich tryptophan (Fig. 1b). Therefore, we set out to explore a site-selective crosslinking strategy to methyllysine readers.

Norleucine-ε-dimethylsulfonium as a methyllysine mimic

By evaluation of molecular interactions between methyllysine peptides and readers from Protein Data Bank (Fig. 1a), we designed norleucine-ε-dimethylsulfonium (NleS⁺me2) as a warhead candidate. First, NleS⁺me2 and Kme2 have the same positive charge and similar molecular shape. As a result, NleS⁺me2 is likely to mimic methyllysine to interact with the reader aromatic cage at the same level. Second, NleS⁺me2 is electron deficient so that it has potential to react with electron-rich indole of tryptophan. Third, sulfonium is active on single-electron transfer (SET) with conjugation capability.

We thus started to develop a general synthetic method to prepare NleS⁺me2-containing peptides. By following the previous literature, L-NleSme-OH (2) was obtained from diethyl acetamidomalonate (1) in 5 steps (Fig. 2a)³¹. After Fmoc protection of amine, compound 3 was obtained as a building block of solid-phase peptide synthesis (SPPS), that can be incorporated at specific site during peptide synthesis³². The cleaved and purified sulfide-containing peptide was converted to sulfonium via S-methylation by iodomethane³³. We first synthesized histone H3K9NleS⁺me2(1–15) peptide (5) for the following study (Fig. 2a).

Next, we prepared recombinant chromodomain of CBX1 (chromobox protein homolog 1) to evaluate activities of sulfonium peptide 5 because CBX1 is a well-studied reader of histone H3K9 methylation for heterochromatin formation (Fig. 2b, Extended Data Fig. 1a)³⁴. Isothermal titration calorimetry (ITC) data demonstrated that H3K9NleS⁺me2 binds to CBX1 at comparable level of H3K9me2 (Fig. 2c). The clear binding activity encouraged us to attempt crosslinking between sulfonium and the tryptophan inside binding pocket. From the reported reactivity of nucleophilic indole and electrophilic sulfonium³⁵, we initially considered that indole may attack sulfonium through S_N2 substitution, but no desired product was observed from reaction screening. Instead, we switched to SET process from electronically excited indole^20,36. Based on the absorbance spectrum of tryptophan, a UV-B lamp with a 305 nm longpass filter (Extended Data Fig. 2a) was used for irradiation of tryptophan. A mixture of CBX1 and H3K9NleS⁺me2 peptide in ice bath was exposed to UV and the crude mixture at different time points was analyzed by UPLC-Q-TOF-MS. The mass spectra revealed two conjugate products with full conversion after 30 min (Extended Data Fig. 3b). Deconvolution mass peaks indicated the products are a methyl adduct and a peptidyl adduct with a loss of dimethyl sulfide (Fig. 2d). Similar chemical transformation was observed between peptide 5 and recombinant MPP8 (Extended Data Fig. 1b and 4) as another chromodomain reader of H3K9 methylation³⁷. Top-down mass spectrometry analysis of the two CBX1 conjugates demonstrated that the peptidyl or methyl group was added to Trp42 (Fig. 2e and Extended Data Fig. 3e), that is in the methyllysine binding pocket (Fig. 1a and 2b).

Mechanism for photo crosslinking of NleSme2

With the initial success of crosslinking, we decided to investigate the reaction using H3K9NleS⁺me2 peptide and CBX1. Since the conjugate was on Trp42 rather than Trp52 (Fig. 2e), we proposed the selectivity is due to a binding mediated crosslinking between sulfonium and the reader (Fig. 3a). We carried out two experiments with additional guanidium chloride for denaturing CBX1 or additional H3K9me3 peptide for binding competition, and the sulfonium lost the reactivity to CBX1 (Fig. 3b). The data demonstrated that the crosslinking is dependent on the association between CBX1 and the sulfonium peptide. To validate the importance of light, we carried out an experiment with successive UV-B irradiation and the conversion process demonstrated that the conjugation was light dependent (Fig. 3d). UV-A light failed to induce the reaction so the excitement of indole by UV-B is a key factor (Fig. 3c).

To further understand the reaction mechanism, we conducted kinetic analysis of the correlation between initial reaction rate V₀ and peptide concentration. The V₀ exhibited saturation with the increase of peptide concentration in the presence of 10 µM CBX1, and the V_max (Fig. 3e) was 1.1 µM/min. If we apply Michaelis-Menten equation for analysis, the calculated K_m is 18 µM, that is close to the measured K_d value of H3K9NleS⁺me2 and CBX1(13 µM, Fig. 2c). This further confirms that the formation of a protein-ligand (PL) complex is a key step of crosslinking. Next, we focused on the kinetics of the photo crosslinking from the PL complex. The bio-layer interferometry (BLI) binding assay revealed the binding rate was very fast (Extended Data Fig. 3a) so 15 min preincubation time is sufficient to reach equilibrium of the PL complex. We added high concentration of sulfonium peptide for saturation so the concentration of CBX1 is almost equal to the concentration of the PL complex. As a result, we could calculate the rate constant k from the plot of V₀ and CBX1 concentration (Fig. 3f). The correlation appeared as a first order reaction, so the crosslinking reaction is likely to happen inside the CBX1-peptide complex, similarly to an intramolecular process³⁸.

With all the data above, we proposed that the NleS⁺me2 peptide first binds to the reader via aromatic cage, and subsequently accepts an electron from excited indole by UV-B, followed by formation of alkyl-tryptophan conjugate and sulfide through SET. In one case, peptidyl conjugate is the product with a leave of dimethyl sulfide (Fig. 3g) and in another case, methyl conjugate and H3K9NleSme are yielded. We hypothesize that SET from excited indole to sulfonium in the pocket contributes to the high reactivity and site-selectivity. When the Trp dispersed in solvent, relaxing channel is much faster than donor-acceptor transfer channel, so that the conjugation rarely happens, like the case with guanidium chloride. When the two molecules are bound together, the more rigid geometry enables to decrease the reorganization energy between excited and ground states of tryptophan so that the relaxing channel is suppressed. Moreover, diabatic coupling strength between tryptophan and sulfonium will be increased so that donor-acceptor transfer channel becomes broadening. Therefore, donor-acceptor electron transfer is more accessible when two groups are bound together in the aromatic cage. We characterized UV absorption of the mixture of peptide and CBX1 that is slightly higher than CBX1 in UV-B region without red-shift (Extended Data Fig. 2b). The minimal difference in absorption is possibly due to the σ-π electron-donor-acceptor (EDA) interaction other than classic π-π EDA interaction. The radical scavenger TEMPO (2,2,6,6-tetramethylpiperidine 1-oxyl) barely reduced the conversion of CBX1 (Extended Data Fig. 3c,d), that may be caused by fast radical transfer inside the pocket with negligible exposure to the spin trap. In sum, the mechanistic study revealed how the binding-mediated crosslinking is highly site-selective to tryptophan inside reader binding pockets with potential for complicated biological samples.

NleSme2-tryptophan crosslinking is site-selective

Since aromatic cage is a general reader strategy to bind methyllysine, the NleS⁺me2-mediated crosslinking is expected to be applicable broadly. We hence synthesized histone H3K4NleS⁺me2 and H4K20NleS⁺me2 peptides, and prepared multiple types of reader domains (Extended Data Fig. 1c-f) including recombinant BPTF (PHD domain)³⁹, JMJD2A (Tudor domain)^40,41, mORC1 (BAH domain)¹² and dSfmbt (MBT domain)⁴². Under our expectation, each reader was readily conjugated by the corresponding histone NleS⁺me2 peptides (Fig. 4a, Extended Data Fig. 6). In addition, non-specific crosslinking to tryptophan that is not in a proper pocket for NleS⁺me2 binding is expected to be negligible based on the reaction mechanism. We applied H3K9NleS⁺me2 peptide to a short tryptophan-containing peptide as well as proteins that contain multiple tryptophan residues including bovine serum albumin (BSA), lysozyme, myoglobin, and cytochrome c. None of them appeared any conjugation product under the standard reaction condition (Fig. 4b, Extended Data Fig. 5). Since the sulfonium-mediated crosslinking is highly site-selective to tryptophan inside specific aromatic cages, we further characterized the selectivity of the histone sulfonium peptides to different readers. BPTF, CBX1, and mORC1 were treated to each histone NleS⁺me2 peptide, and the reactivities appeared very selective (Fig. 4c).

Methyllysine readers generally prefer to bind a specific methylation state, that is an important mechanism for different biological roles of Kme1, Kme2, and Kme3. But most known readers are not exclusively to one methylation state, and therefore, we expected that NleS⁺me2 is active to crosslink diverse readers as a Kme2 mimic. We thus set up crosslinking reaction to dSfmbt (prefers Kme1)⁴², mORC1 (prefers Kme2)¹², and BPTF (prefers Kme3)³⁹. All the readers were readily crosslinked by the sulfonium peptides, and the reduced amount of conjugation by excessive Kme1, Kme2, and Kme3 matched the binding preference of methylation state (Fig. 4d). As a result, NleS⁺me2 based probes are capable of broad methyllysine reader studies.

We started this work with NleS⁺me2 because it was designed as a structural mimic of Kme2. With the success of crosslinking to methyllysine readers, we were curious about activities of sulfonium based on other methionine homologues. Therefore, we additionally synthesized histone peptides with norvaline-dimethylsulfonium (NvaS⁺me2) and S-methylmethionine (Met⁺me) to compare the crosslinking activities (Fig. 4e, Extended Data Fig. 6). The three sulfonium showed similar reaction rate to CBX1, while NleS⁺me2 was faster than the other two to BPTF. In the case of dSfmbt, NleS⁺me2 was the only active sulfonium to crosslink. The different rate of peptidyl conjugate formation is likely due to tryptophan position in aromatic cage (Fig. 1a). CBX-W42 and BPTF-W2891 are proximate to lysine γ-CH₂ and δ-CH₂ so shorter sulfonium still can accept electron from excited indole via SET. However, dSfmbt-W944 is close to ε-methylamine so that shorter sulfonium cannot reach W944 for crosslinking. Consequently, we think NleS⁺me2 is an optimal sulfonium to target methyllysine readers broadly.

Because tryptophan-containing aromatic cage is a general strategy for affinity to methyllysine, the sulfonium-based probe has potential to crosslink binding proteins without known 3D structures. Commercial antibodies are important tools to identify site-specific methyllysine, but the sequence and 3D structure are not disclosed. We picked a H3K4me3 and a H3K9me3 antibody and the data demonstrated that the conjugations were selective by the corresponding histone NleS⁺me2 peptides (Fig. 4f-g).

Investigating nuclear reader proteins using NleS⁺me2 probes

Encouraged by the reactivity and selectivity to recombinant readers and antibodies in vitro, we next moved to complicated cell samples. H3K4NleS⁺me2 peptide with a desthiobiotin tag was added to extracted nuclei of HeLa cells followed by UV-B irradiation for 5 min in the presence of additional unmodified (Group I) or H3K4me3 peptides (Group II) for competition (Fig. 5a). After washing and sonication of the nuclei, the peptidyl conjugates were enriched by streptavidin resin and analyzed by LC-MS/MS. The comparison between the two groups was illustrated as volcano plot (Fig. 5b), and methyllysine dependent binding proteins were expected to appear in the top right area. Consequently, most reported H3K4 methylation readers^7,43,44 were identified unambiguously in the plot. We carried out another experiment using H3K9NleS⁺me2 probe and the volcano plot also exhibited the majority of known readers (Extended Data Fig. 7a)^7,44,45. In addition to the known readers, many protein hits are in large protein complexes that contain methyllysine readers according to literature studies. For example, KAT5(TIP60), MEAF6, and EPC1/2 associate with reader ING3 in NuA4 complex, and BRPF1-3, MEAF6, and KAT7 associate with reader ING5 in MORF complex⁴⁶. Also, ASH2L, RBBP5, DPY30, BAP18, and WDR5 are parts of MLL complex that associates H3K4me3 via KMT2A^47,48. These hits were probably enriched from tight protein-protein interactions with crosslinked readers. The results demonstrated that sulfonium probes are capable of selective crosslinking to various types of methyllysine binding proteins in intricate proteome.

Since the crosslinking is at specific residues, it is possible to map crosslinked tryptophan from proteome by crosslinking mass spectrometry (XL-MS)⁴⁹. We first prepared several crosslinking mixtures of recombinant CBX1, BPTF, JMJD2A, and mORC1 with sulfonium peptides to develop a protocol. Crosslinked proteins were enriched by streptavidin resin and subsequently digested by GluC protease. After washing, crosslinking peptides were released from resin by trypsin digestion for LC-MS/MS analysis (Fig. 5c, Extended Data Fig. 8). By data search, all the reader fragments that were crosslinked at key tryptophan were clearly identified (Supplementary Table 1 and Supplementary mass spectra). We next applied nuclei samples that were treated by H3K4NleS⁺me2 or H3K9NleS⁺me2 probe, and we were able to identify many crosslinking peptides of known readers with excellent quality of spectra (Supplementary Table 1 and Supplementary mass spectra).

Inspired by XL-MS of known readers, we started to search crosslinking sites of other proteins in the volcano plot (Fig. 5b) with a threshold for high confidence (Supplementary Table 2). We found that BRWD1 and BRWD3 have multiple crosslinked tryptophan that may contribute to methyllysine binding. According to literature studies, BRWD1 and BRWD3 research mainly focused on bromodomain that binds to acetylated histone^50,51. Although PHIP/BRWD2 was reported as an H3K4me3 binding protein⁵², the activity of BRWD3 is not clear since BRWD3 biological roles are different from BWRD1 and BRWD2⁵¹. We thus selected BRWD3 for investigations at molecular level. Because crosslinked W1062, W1063 and W1100 (Fig. 5d and Supplementary mass spectra) are closer to bromodomain than WD domain, we expressed recombinant BRWD3(922–1443) and demonstrated that it can be crosslinked by H3K4NleS⁺me2 peptide (Fig. 5e). On the contrary, the W1062A/W1063A mutant or BRWD3(1140–1443) as deletion mutant lost crosslinking activity dramatically (Fig. 5e). Next, we set up chemical crosslinking experiments and identified clear binding preference of BRWD3 to H3K4me3 than unmodified peptides (Fig. 5f). The results of mutants matched the data of crosslinking activities (Fig. 5e). Although XL-MS identified W1100 as a crosslinking site (Supplementary mass spectra), W1100A mutant did not alter BRWD3 activity (Extended Data Fig. 7b,c). Therefore, the crosslinking at W1100 might be nonspecific due to complicated cellular environment. By AlphaFold prediction, we found W1063 is close to W1089 that may contribute to H3K4me3 binding but W1100 is not likely in an aromatic cage for methyllysine binding (Extended Data Fig. 7d). We next prepared HeLa nuclei with transiently expressed 3xFLAG-BRWD3(922–1450) for crosslinking by H3K4NleS⁺me2. The BRWD3-conjugate was more selectively reduced by H3K4me3 peptide competition than H3 unmodified peptide (Fig. 5g). In addition, 3xFLAG-BRWD3 from nuclear extracts was selectively pulled down by H3K4me3 peptide (Fig. 5h). All the data demonstrated that BRWD3 is a reader of H3K4me3, and it could explain how BRWD3 recruit KDM5 to maintain H3K4 methylation levels⁵³. By the case of BRWD3 study, we could conclude that the sulfonium-based probe could map proteins and specific tryptophan that interact with methyllysine peptides with broad application potential.

Applicability of sulfonium-tryptophan crosslinking

Based on the investigation of crosslinking between NleS⁺me2 peptide and methyllysine readers, we envision that the reaction scope could be expanded to any tryptophan with nearby sulfonium interactions. We firstly consider expanding reaction scope to alkylamine binding proteins beyond methyllysine readers. Betaine and choline are important metabolites with quaternary amine, that bind to specific proteins via aromatic cage with tryptophan (Fig. 6a). Hence, we synthesized sulfonium analogues of betaine and choline to characterize the crosslinking activities (Fig. 6b). Under our expectation, sulfonium 9 was active to crosslink ProX and OpuAC^54,55, and the reactivity could be diminished by additional betaine (Fig. 6c, Extended Data Fig. 9). Similar result was found from sufonium 11 and ChoX⁵⁶ (Fig. 6c). In addition, we tested reactivities of sulfonium analogues of acetylcholine and SAM, but none of them were active to crosslink the betaine and choline binding proteins (Extended Data Fig. 9). The orthogonal results further demonstrated the reactivity and selectivity of the sulfonium-tryptophan crosslinking that require specific close contact via binding process.

We next consider expanding reaction scope to tryptophan not in an aromatic cage. Proximate contact between sulfonium and tryptophan could be driven by protein-ligand interactions (Fig. 6d). NanoBiT is a split protein complex that is used to study protein-protein interactions⁵⁷. By evaluating the interface between LgBiT and SmBiT, we found W11 locates on LgBiT surface without any aromatic residues around (Extended Data Fig. 10a,b), that is spatially close to SmBiT-E166 (Fig. 6e and Extended Data Fig. 10a,b). We thus synthesized SmBiT peptides with sulfonium residues at E166 for potential crosslinking. Under UV-B irradiation, SmBiT-E166Met⁺me was active to crosslink LgBiT but not to W11F mutant (Fig. 6f). Interestingly, SmBiT-E166NvaS⁺me2 was much less active and SmBiT-E166NleS⁺me2 was inactive to conjugate LgBiT. Based on the interface between W11 and E166, we think the molecular shape of Met⁺me is like glutamate so the sulfonium warhead has the best interaction to LgBiT-W11. However, the extra methylene of NvaS⁺me2 and NleS⁺me2 reduced accessibility of sulfonium to LgBiT-W11, that resulted in much less crosslinking reactivities. Since the crosslinking between LgBiT and SmBiT-E166Met⁺me is highly selective, we proposed it is potentially bioorthognal in complicated cellular environment. We thus applied a sulfonium Myc-SmBiT peptide and LgBiT in HeLa cell lysate for crosslinking. Western blot data demonstrated that LgBiT was crosslinked by the sulfonium SmBiT with high selectivity and the reaction conversion rate was not affected by lysate (Fig. 6g and Extended Data Fig. 10c).

By results above, we could conclude that the photo-induced sulfonium-tryptophan crosslinking is not limited to methyllysine readers. It is generally applicable to develop sulfonium probes to crosslink proximate tryptophan based on specific protein-ligand interactions. Since there are diverse microenvironment of tryptophan in proteome, it has wide application potential to develop new biorthogonal tools in chemical biology.

In summary, we developed a photo crosslinking strategy to site-specific methyllysine readers. Unlike traditional photo crosslinker based on highly reactive intermediate from irradiation, our method relies on electron donor-acceptor interaction driven by specific reader-ligand binding. Sulfonium peptide selectively crosslinks to the excited tryptophan inside aromatic cage under UV-B irradiation. Due to the relaxation of excited tryptophan without proximate sulfonium, the non-specific crosslinking is expected to be minimal. Moreover, laborious optimization of placement of photoreactive group can be avoided since the sulfonium is both a methyllysine mimic and a warhead. Therefore, this method could be widely used to investigate methyllysine readers from cell and tissue samples. Given the reported number of methyllysine sites is growing rapidly from state-of-the-art proteomic study, we hope sulfonium-based tools will accelerate discovery of previously unknown methyllysine readers for comprehensive understanding of lysine methylation from histone to nonhistone. Finally, the crosslinking concept in this study could be expanded to develop more tryptophan site-selective conjugation beyond methyllysine readers, that could be widely applied to bioorganic chemistry and chemical biology study.

General methods

All reagents and chemicals were purchased from Energy Chemical, Macklin, and Bidepharmatech, and used as received without any further purification. Bis-PEG5-NHS ester was purchased from Leyan. All Fmoc-protected amino acids, rink amide resin, and Fmoc-HoMet-OH were purchased from GL Biochem Co., Ltd. (China). Some peptides were purchased from Bankpeptide Biological Technology Co., Ltd (China) including histone methyllysine peptides for sulfonium binding competition, N-FITC (fluorescein isothiocyanate) histone methyllysine peptides for fluorescence polarization assay, H3K4M and H3K9M for Met⁺me peptide preparation, and histone peptides with biotin for chemical crosslinker assay. NMR spectra were recorded on 500 MHz or 600 MHz Bruker BioSpin, Switzerland. UPLC-Q-TOF-MS analyses were performed with G2 XS high resolution mass spectrometer using Waters Acquity UPLC BEH C18 (1.7 µm, 2.1 × 50 mm) or Waters Acquity UPLC Protein BEH C4 (1.7 µm, 2.1 × 50 mm). Linear gradients using A: H₂O (0.1% HCOOH) and B: CH₃CN (0.1% HCOOH) over varying periods of time. Bruker rapiflex MALDI-TOF-MS was used for characterization of peptides. Semi-preparative HPLC was carried out on a Waters 1525 pump with 2489 detector using a XBrigde BEH C18 (10 µm, 19 × 250 mm) column. Linear gradients using A: H₂O (0.1% TFA) and B: CH₃CN (0.1% TFA) over varying periods of time. Peptide centrifugation was performed by highspeed refrigerated micro centrifuge MX-307 purchased from TOMY KOGYO Co., Ltd. Peptide freeze drying was achieved by Labconco FreeZone Benchtop Freeze Dryer. 302 nm or 365 nm light source was performed on a Analytikjena UVP Crosslinker CL-1000. SCHOTT N-WG305 50x50mm 1mm T LP Filter (14466) was purchased from Edmund Optics. BSA (A8020) was purchased from Solarbio. Lysozyme (L6876), myoglolin (M0630), and cytochrome c (C7752) were purchased from Sigma-Aldrich.

Chemical synthesis methods

The detailed synthetic methods, NMR spectra and mass spectra for all the peptides and small molecules are provided in the Supplementary Information.

Recombinant protein expression and purification

The chromodomains from human CBX1 (residues 20-73, C60A) and human MPP8 (residues 55-116) were cloned into pET-21(+) vector with a C-terminal 6×His tag. Proteins were over-expressed in BL21(DE3) Escherichia coli cells by induction of 0.25 mM isopropyl β-D-thiogalactoside at 30 ºC for 3 h when OD₆₀₀ reached 0.6-0.8 in the LB medium. Harvested cells were suspended in lysis buffer (20 mM Tris, 150 mM NaCl, 0.2 mM PMSF, pH 7.5) and then lysed by sonication. The clarified lysate by centrifugation was applied to nickel resin equilibrated in lysis buffer. The resin was washed sequentially by lysis buffer, high salt buffer (20 mM Tris, 500 mM NaCl, pH 7.5) and 20 mM imidazole buffer (20 mM Tris, 150 mM NaCl, 20 mM imidazole, pH 7.5). The target proteins were eluted by 200 mM imidazole buffer (20 mM Tris, 150 mM NaCl, 200 mM imidazole, pH 7.5). Purified proteins in the storage buffer (20 mM Tris, 150 mM NaCl, pH 7.5) were finally obtained after dialysis and concentration. The proteins were snap-frozen and stored at -80 ºC.

The detailed expression and purification procedure of the other recombinant proteins in this study are provided in the Supplementary Information

Isothermal titration calorimetry

ITC experiments were conducted at 25 ºC using MicroCal PEAQ-ITC Automated (Malvern Instruments) by titrating peptides into proteins at 25 ºC. To the chromo domain of CBX1 in 20 mM Tris, 150 mM NaCl, pH 7.5, H3(1-15) peptides containing mono-, di- or tri methylation at K9 and H3K9NleS⁺me2 peptide (S16) (250-1000 μM) in the same buffer were titrated into proteins at 25 μM. A total of 19 injections were performed with 0.4 μL for the first and 2.0 μL for the rest. Each spacing was 150 s and the reference power was 10 μcal/s. Data was modeled using the “One Set of Sites” supplied in MicroCal PEAQ-ITC Analysis software (version 1.30). The resultant ITC curves were processed using GraphPad Prism software.

Sulfonium-mediated crosslinking to methyllysine readers

General Crosslinking Procedure. Reader protein in stock solution was diluted by HEPES buffer (100 mM HEPES (pH=7.5), 10 mM glutathione) and mixed with sulfonium peptides in ice bath for 15 min. The solution (total volume: 50 μL) was transferred into a 96-well plate and irradiated for 5 min in ice bath using a 302 nm UV lamp (under 305 nm long-pass filter). The reaction mixture was later analyzed by UPLC-Q-TOF-MS with a gradient of 5-95% B over 4 min and 95% B over 2 min. The analytic yield was calculated based on mass peak areas of starting material and products from deconvolution of the mass spectrometry data. Yield = A_p/A_s where A_p is the peak area of peptide-conjugated product and A_s is sum of all protein peak areas including the residual starting material, peptidyl product, methyl products, and other side product (if any).

Kinetics study of the crosslinking between H3K9NleS⁺me2 and CBX1.

10 μM CBX1 in HEPES buffer (100 mM HEPES, pH=7.5) was mixed with H3K9NleS⁺me2 peptide (5) of serial concentrations. All the samples were incubated on ice for 15 min and then transferred into a 96-well plate for 3 min photo crosslinking as described in General Crosslinking Procedure. According to integration of mass peaks from UPLC-Q-TOF-MS analysis, the initial reaction rate V₀ was calculated from analytic yield of peptide-CBX1 conjugate. Finally, the processed data were fitted by the program GraphPad Prism (equation: Michaelis-Menten model). Next, CBX1 at different concentrations in HEPES buffer was mixed with 625 μM H3K9NleS⁺me2 peptide (5). After photo crosslinking at the same condition, the resulting mixtures were analyzed by UPLC-TOF. The data were processed by the program GraphPad Prism (equation: first order polynomial model (straight line)).

Investigation of nuclear methyllysine readers by sulfonium probes.

HeLa cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco, C11965500CP) containing 10% fetal bovine serum (FBS), 10 U/m penicillin and 100 mg/mL streptomycin (HyClone, SV30010) at a humidified 37 ºC incubator with 5% CO₂. After cells in ~90% confluency were harvested, the in situ crosslinking experiments were carried out referred to reported methods with some modifications^58,59. Briefly, 5×10⁶ Hela cells were lysed in hypotonic lysis buffer A (10 mM Tris, 15 mM NaCl, 1.5 mM MgCl₂, 0.2 mM PMSF, pH 7.6) for 10 min on ice, then homogenized with 5 strokes of a loose pestle Dounce homogenizer. The nuclei were isolated by spinning 200 g for 5 min at 4 °C, and resuspended in crosslinking buffer (100 mM HEPES, 1.5 mM MgCl₂, 150 mM KCl, 0.2 mM PMSF, pH 7.5). Next, sulfonium peptide probe (50 μM) and unmodified peptide (500 μM) were added into the isolated nuclei in group I. The same amount of sulfonium peptide and Kme3 peptide were added to group II nuclei. The mixture was incubated for 20 min followed by irradiation with UV-B light for 5 min on ice. After that, the excess peptides were removed via nuclei washing with crosslinking buffer twice. Next, the nuclei were resuspended with extraction buffer (25 mM HEPES, 1.5 mM MgCl₂, 300 mM KCl, 1 mM EDTA, 0.1% NP40, 0.5 mM DTT, pH 7.5), were sheared by sonication with 8 cycles of a probe sonicator at 20% amplitude for 5 s on and 10 s off. After the removal of any insoluble materials by centrifugation, the nuclear proteins were loaded onto pierce streptavidin magnetic beads (Thermo Fisher, 88817), which were blocked and equilibrated with 1 mg/mL BSA and extraction buffer. The subsequent immunoprecipitation was carried out at 4 °C for 2 h with end-over-end rotation.

The beads were sequentially washed with extraction buffer twice, high salt buffer (25 mM HEPES, 1 M NaCl, pH 7.5), urea buffer (25 mM HEPES, 2 M urea, pH 7.5), TE buffer (25 mM HEPES, 1 mM EDTA, pH 7.5) and 50 mM ammonium bicarbonate at pH 8.0 twice. Next, trypsin Gold (2 μl of a 1 μg/μL stock) was added to the beads in 200 μl ammonium bicarbonate, and the on-beads digestion was performed overnight at 37 °C with 1500 rpm in a thermo mixer (IKA Matrix Orbital). Additional trypsin (1 μL of a 1 μg/μL stock) was added for another 2 h digestion. The supernatant was collected, and the beads were washed with ammonium bicarbonate. The combined supernatants were lyophilized.

The lyophilized samples were resuspended in 15 μL 0.1% formic acid for LC-MS/MS analysis. The peptides were separated by a 120 min gradient elution at a flow rate 0.300 µL/min with the Thermo Vanquish Neo integrated nano-HPLC system which is directly interfaced with the Thermo Exploris 480 mass spectrometer. The analytical column was a home-made fused silica capillary column (75 µm ID, 150 mm length; Upchurch, Oak Harbor, WA) packed with C-18 resin (300 A, 3 µm, Varian, Lexington, MA). Mobile phase A consisted of 0.1% formic acid in water, and mobile phase B consisted of 80% acetonitrile and 0.1% formic acid. The mass spectrometer was operated in the data-dependent acquisition mode using the Xcalibur 4.1 software and there is a single full-scan mass spectrum in the Orbitrap (350-1800 m/z, 60,000 resolution) followed by 20 data-dependent MS/MS scans at 30% normalized collision energy. The AGC target was set as 5e4, and the maximum injection time was 50 ms. Each mass spectrum was analyzed using the Thermo Xcalibur Qual Browser and Proteome Discoverer for the database searching against the Mus musculus proteome database downloaded from UniProtKB (UP000000589) containing 55,311 proteins and the Homo sapiens proteome database downloaded from UniProtKB (UP000005640) containing 80,581 proteins as of October 18, 2022, respectively. The Sequest search parameters included a 10 ppm precursor mass tolerance, 0.02 Da fragment ion tolerance, and up to 2 internal cleavage sites. Fixed modifications included cysteine alkylation, and the methionine oxidation was variable modification. Peptides were filtered with 1% false discovery rate (FDR). These values were subsequently adjusted for two-tail t-test. Protein ratios with a ratio greater than 2.0 and a P-value less than 0.05 were considered significant. The mass spectrometry data have been deposited in the ProteomeXchange Consortium repository as an open-source dataset under the identifier PXD051693.

Crosslinking mass spectrometry (XL-MS) of peptidyl conjugate by sulfonium probes.

XL-MS of recombinant readers protein. Crosslinking mixtures of BPTF with H3K4NleS⁺me2 peptide (S14), JMJD2A with H3K4NleS⁺me2 peptide (S14), JMJD2A with H4K20NleS⁺me2 peptide (S15), mORC1 with H4K20NleS⁺me2 peptide (S15), CBX1 with H3K9NleS⁺me2 peptide (S16) were reduced with 10.0 mM dithiothreitol at 37°C for 1 hour, followed by alkylation with 20 mM iodoacetamide in an aqueous solution for 30 minutes at room temperature in the dark. The beads were then washed three times with 50 mM ammonium bicarbonate at pH 8.0. Digestion was performed with Glu-C at an enzyme-to-protein ratio of 1:30 (w/w) overnight at 37°C. Afterward, the beads were washed twice with 50 mM ammonium bicarbonate. Subsequently, 10 μL of the sample was digested using a mixture of Lys-C and trypsin enzymes at enzyme-to-protein ratios of 1:50 (w/w) and 1:30 (w/w) respectively, at 37°C for 10 hours. Finally, the sample was desalted using homemade Venusil XBP C18 (5 μm, 150 Å) desalting tips prior to LC-MS/MS analysis.

The sampless were initially re-dissolved in a solution containing 0.1% formic acid (FA). These samples were analyzed using an Easy-nLC 1000 system coupled with a Q-Exactive mass spectrometer (Thermo Fisher Scientific, USA). The control of the mass spectrometer and data collection were managed using the Q-Exactive Tune Application (2.8 SP1 Build 2806) and Thermo Scientific Xcalibur software (v3.1.66.10), respectively. The samples were automatically loaded onto a C18 reversed-phase (RP) trap column (150 μm i.d. × 3 cm) and separated on a C18 capillary column (150 μm i.d. × 15 cm), which was in-house packed with ReproSil-Pur C18-AQ particles (1.9μm, 120 Å). The mobile phases used were buffer A (98% H₂O, 2% ACN, 0.1% FA) and buffer B (2% H₂O, 98% ACN, 0.1% FA). The separation gradient was programmed as follows: 2–10% B over 10 min, 10–23% B over 50 min, 23–40% B over 20 min, 40–80% B over 2 min, followed by a hold at 80% B for 13 min. The mass spectrometry settings included data-dependent acquisition, full MS resolution of 70,000 at m/z 200, a scan range of 300–1800, MS1 automatic gain control (AGC) target of 3e6, MS1 maximum injection time (IT) of 60 ms, MS/MS resolution of 17,500 at m/z 200, a fixed first mass of 110 m/z, MS/MS AGC target of 5e4, MS/MS maximum IT of 60 ms, a loop count of 20, an isolation window of 2.0 m/z, higher-energy collision dissociation (HCD) with a normalized collision energy (NCE) of 28, charge exclusion for unassigned, 1, and >8 charges, an intensity threshold of 1000, and a dynamic exclusion of 18 s.

XL-MS of nuclei samples. Swelled 3.5x10⁶ HeLa cells with 12 mL 1xRSB buffer and incubated on ice for 15 min. Collected crude nucleus by 4 ºC, 200 g, 5min. Leave about 1.5 mL supernatant to resuspended pellet and homogenized with 5 strokes. Then, nucleuses were pelleted at 4 ºC, 200g for 5min, discarded supernatant and resuspended nucleuses with crosslinking buffer (100 mM HEPES, pH 7.5, 150 mM KCl, 1.5 mM MgCl₂, added0.2 mM PMSF before use). The nuclei were incubated with sulfonium probes (50 μM) and incubated on ice for 15 min followed with irradiation (302 nm UV light, 305 nm filter, ice, 5 min). The un-crosslinked probes were removed by twice crosslinking buffer washing (4 ºC, 200 g, 5 min). The nuclei were resuspended in extraction buffer (25 mM HEPES, 1.5 mM MgCl₂, 300 mM KCl, 1 mM EDTA, 0.1% NP40, 0.5 mM DTT, pH 7.5), and nuclear proteins were extracted through sonication, using 8 cycles of a probe sonicator at 20% amplitude for 5 seconds on and 10 seconds off. Following centrifugation to remove any insoluble materials, the nuclear protein was loaded onto Pierce high-capacity streptavidin agarose resin (Thermo Fisher, 20359) and immunoprecipitated at room temperature for 3 hours with end-over-end rotation.

The beads were washed sequentially three times with extraction buffer and twice with 50 mM ammonium bicarbonate. Next, 10.0 mM dithiothreitol was added and incubated at 37°C for 1 hour, followed by the addition of a 20.0 mM iodoacetamide aqueous solution, which was incubated at room temperature for 30 minutes in the dark. After centrifugation, the beads were washed twice with 50 mM ammonium bicarbonate. Glu-C (30 μg) was then added to the beads in 600 μL ammonium bicarbonate, and on-beads digestion was performed overnight at 37°C. The beads were washed with 50 mM ammonium bicarbonate twice. Then additional Lys-C (15 μg) and trypsin Gold (30 μg) were added, and digestion continued at 37°C for another 10 hours. After digestion, the supernatant was collected, and the beads were washed twice with ammonium bicarbonate and twice with 20% acetonitrile in water, with the wash solutions also being collected. The combined supernatants were then lyophilized.

For peptide, homemade C18 tips (5 μm, 100 Å; Durashell) were employed for desalting and fractionation. After activating and equilibrating the C18 tips, the peptides were loaded onto the tips and washed three times with solvent A (H₂O with ammonia added, pH 10.0) for desalting. The elution solvent, solvent B (acetonitrile with ammonia added, pH 10.0), was used to create 9 eluates (6%, 9%, 12%, 15%, 18%, 21%, 25%, 30%, 80% B), which were combined into fractions as follows: 6% and 25% for fraction 1, 9% and 30% for fraction 2, 12% and 80% for fraction 3, and 15%, 18%, and 21% for fractions 4, 5, and 6, respectively. Finally, the fractionated peptides were lyophilized and subsequently analyzed using nano-LC-MS/MS.

The samples were re-dissolved in a solution containing 0.1% formic acid (FA) and analyzed using an Easy-nano LC 1200 system, coupled to an Orbitrap Exploris 480 instrument equipped with a FAIMS Pro device (Thermo Fisher Scientific). During FAIMS separations, temperatures of the inner and outer electrodes were maintained at 100 °C, and the total carrier gas flow rate was set to 4.0 L/min. Compensation voltage (CV) values for each injection were -45 and -65. The mass spectrometry analysis utilized two mobile phases: mobile phase A (0.1% FA in HPLC-grade H₂O) and mobile phase B (acetonitrile with 20% H₂O and 0.1% FA). Peptides were separated on a C18 capillary column (150 μm i.d. × 150 mm) packed with C18 silica particles (1.9 μm, 120 Å) from Dr. Maisch GmbH, with the column heated to 55°C and a flow rate of 600 nL/min. The gradient started at 5% B, increasing to 9% B over 10 minutes, then from 9% to 20% B over the next 35 minutes, followed by an increase from 20% to 35% B over 40 minutes, and finally from 35% to 48% B over 25 minutes. The mass spectrometer operated in positive ion mode with data-dependent acquisition (DDA). MS1 scans were performed at a resolution of 60,000 (at m/z 200) from m/z 350 to 1500, and MS2 scans at a resolution of 15,000. The maximum injection times were set to 20 ms for MS1 and 30 ms for MS2. In each full MS scan, the most intense ions with charge states from 3 to 7 were selected for sequencing, using an isolation window of 1.6 m/z and a cycle time of 2 seconds. Fragmentation of precursor ions was achieved using HCD mode with a normalized collision energy of 30.

Mass data analysis. For the analysis of crosslinked peptides, the raw files were processed using the OpenUaa software⁶⁰, with searches conducted against the protein FASTA file of model readers protein and quantitative proteomics-derived readers. Carbamidomethyl (C) was considered as fixed modification; Oxidation (M) and acetylation of the protein N-terminus were considered as variable modification. The maximum number of missed cleavage sites was set to three. The precursor mass tolerance and fragment mass tolerance were both set at 20 p.p.m. The search results were filtered using a false discovery rate (FDR) of 5% at the peptide-spectrum match (PSM) level. The mass spectrometry data have been deposited in the ProteomeXchange Consortium repository as an open-source dataset under the identifier PXD049149.

Characterization of BRWD3 as a H3K4me3 reader

Crosslinking between recombinant BRWD3 and H3K4NleS⁺me2 peptide. 3μM GST-tagged BRWD3 (922-1443), BRWD3 (922-1443_W1062A&W1063A), BRWD3 (922-1443_W1100A), or BRWD3 (1140-1443) was mixed with H3K4NleS⁺me2 peptide (S14) (100 μM) for 10 min crosslinking by General Crosslinking Procedure. The crosslinking mixtures were analyzed by western blot using pierce™ high sensitivity streptavidin-HRP, and total proteins were analyzed using α-GST antibody.

Analysis of BRWD3-H3K4me3 peptide interaction by chemical crosslinker. 4 μM putative reader protein with GST tag was mixed with 50 μM H3K4me3 or unmodified H3 peptide with biotin tag. The mixture was incubated on ice for 20 min before adding 1 mM chemical crosslinker (Bis-PEG5-NHS ester). The crosslinking reaction was allowed to proceed for 20 seconds at room temperature, followed by quenching using 100 mM Tris for another 15 min. The resulting sample was analyzed by western blot using HRP-conjugated Streptavidin and α-GST antibody respectively.

Crosslinking BRWD3 in nuclei by H3K4NleS⁺me2 peptide. Human BRWD3(922-1450) was cloned into pcDNA3.1 expression vector with an N-terminal 3xFLAG tag. Endofree plasmid was prepared by following manufacturer’s instruction (CWBIO, CW2107M). HeLa cells were seeded in 100 mm dishes with about 40% confluent one day before and were later transfected with the plasmid when growth to 70-80% confluent. 15 μg plasmid was diluted by 250 μL Opti-MEM medium and mixed with 30 μL P3000. Next, 12 μL lipo3000 in 250 μL medium was added and incubated for 15 min at room temperature. The about 500 μL plasmid-lipid complex solution was later added to 10 mL HeLa cell culture in 100 mm dish for BRWD overexpression. After 24h, the cells were harvested for following crosslinking reactions.

1x10⁶ HeLa cells were swelled with 4 mL 1xRSB buffer (10 mM Tris-HCl, pH 8.0, 15 mM KCl, 1.5 mM MgCl₂, fresh0.2 mM PMSF) and incubated on ice for 15 min. Crude nuclei were collected by 200 g centrifugation at 4 ºC and resuspended with 1.5 mL buffer for homogenization with 2 strokes. The nuclei were next pelleted and resuspended with crosslinking buffer (100 mM HEPES, pH 7.5, 150 mM KCl, 1.5 mM MgCl₂, fresh 0.2 mM PMSF). The nuclei were incubated with unmodified peptide (500 μM) or Kme3 peptide (500 μM) on ice for 8 min. Next, sulfonium probe S14 (50 μM) was added into each tube for 15 min incubation on ice. After 5 min photo crosslinking, the remaining peptides were removed by twice washing with crosslinking buffer. Next, the nuclei were resuspended with sonicate buffer (25 mM HEPES, pH 7.5, 1.5 mM MgCl₂, 300 mM KCl, 1 mM EDTA, 0.1% NP40, fresh 0.5 mM DTT) for subsequent sonication (5 s on, 10 s off, 8 cycles, AMP: 25%). Centrifugation (4 ºC, 13000 g, 10 min). After centrifugation, the nuclear protein was loaded onto pierce streptavidin magnetic beads (Thermo Fisher, 88817), which were equilibrated and blocked with extraction buffer and 1 mg/mL BSA. The immunoprecipitant was carried out at 4 °C for 2 h with end-over-end rotation. The protein bound beads were washed with sonicate buffer, high salt buffer (50 mM Tris, 1M KCl, pH 7.5), urea buffer (50 mM Tris, pH 7.5, 2M urea), EDTA buffer (50 mM Tris, pH 7.5, 1 mM EDTA), Tris-HCl buffer (50 mM Tris, pH 7.5) and 1xPBS buffer for one time. Next, the beads were boiled with 40 μL 1xSDS buffer at 95 ºC for 30 min and the enriched proteins were analyzed by western blot.

Immunoprecipitation (IP). Streptavidin magnetic beads (500 μg) were preincubated with biotin-H3Kme0 peptide (1.2 nmol) or biotin-H3Kme3 peptide (1.2 nmol) at 4 °C for 2 h with end-over-end rotation. The peptide bound beads were washed with sonicate buffer, urea buffer and sonicate buffer twice. Next, nuclei were extracted from HeLa cells with BRWD overexpression and sheared by sonication by the same procedure above. The soluble nuclear proteins were equally separated into peptide bound beads. The immunoprecipitations were carried out at 4 °C for 2 h with end-over-end rotation. The protein bound beads were washed with sonicate buffer, high salt buffer, EDTA buffer, and 1xPBS buffer twice. Next, the beads were boiled with 40 μL 1xSDS buffer at 95 ºC for 30 min and enriched proteins were analyzed by western blot.

Methods-only references

58. Yang, Q., Gao, Y., Liu, X., Xiao, Y. & Wu, M. A general method to edit histone H3 modifications on chromatin via sortase-mediated metathesis. Angew. Chem. Int. Ed. 61, e202209945 (2022).

59. Burton, A. J., Haugbro, M., Gates, L. A., Bagert, J. D., Allis, C. D. & Muir, T. W. In situ chromatin interactomics using a chemical bait and trap approach. Nat. Chem. 12, 520-527 (2020).

60. Liu, C., Wu, T., Shu, X., Li, S. T., Wang, D. R., Wang, N. et al. Identification of protein direct interactome with genetic code expansion and search engine OpenUaa. Adv. Biol. 5, e2000308 (2021).

Competing interests

The authors declare no competing interests.

Author contributions

F.F. synthesized and characterized small molecules and peptides. Y.G. and F.F. prepared recombinant reader proteins and conducted crosslinking analysis. Y.G. carried out reader binding assays and crosslinking kinetic analysis. Q.Z., N.Z. and L.Z. designed and performed crosslinking mass spectrometry. T.L. prepared recombinant betaine and choline binding proteins. T.L. and Q.Y. performed cell-based experiments. Q.Y. studied crosslinking of antibodies. Y.X. conducted chemical crosslinker assay of BRWD3. Y.X. and Y.H. designed and performed NanoBiT crosslinking experiment. J.P. and S.F. conducted 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 figures, Methods, Supplementary Information and commented on the paper.

Acknowledgments

We acknowledge the support from 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 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 Dr. Yinjuan Chen of ISCMS for data acquisition and analysis of sulfonium compounds by mass spectrometry and thank Dr. Zhong Chen of ISCMS for characterization of UV light sources. We also thank 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 Instrumentation and Service Center for Physical Sciences for supporting ITC measurement. We thank Dr. Yu Wang at Westlake University for helpful discussion of the crosslinking reaction mechanism. We thank Dr. Sihui Ma for the assistance with recombinant BPTF preparation.

Data availability

Data that support the findings of this study are available in the Article, Supplementary Information and Source Data. Raw proteomics data are deposited on PRIDE with accession number PXD051693 and PXD049149.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

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SupplementarySpreadsheet.xlsx
Supplementary Data Set
SupplementaryInformation.pdf
ExtendedDataFig.1.jpg
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 three measurements.
ExtendedDataFig.2.jpg
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).
ExtendedDataFig.3.jpg
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.
ExtendedDataFig.4.jpg
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
ExtendedDataFig.5.jpg
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.
ExtendedDataFig.6.jpg
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.
ExtendedDataFig.7.jpg
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.
ExtendedDataFig.8.jpg
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.
ExtendedDataFig.9.jpg
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.
ExtendedDataFig.10.jpg
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.

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

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Journal Publication

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Abstract

Figures

Introduction

Results and Discussion

Norleucine-ε-dimethylsulfonium as a methyllysine mimic

Mechanism for photo crosslinking of NleSme2

NleSme2-tryptophan crosslinking is site-selective

Investigating nuclear reader proteins using NleS⁺me2 probes

Applicability of sulfonium-tryptophan crosslinking

Conclusions

Methods

Declarations

Competing interests

Author contributions

Acknowledgments

Data availability

Reporting summary

References

Additional Declarations

Supplementary Files

Status:

Journal Publication

Version 1

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

Status:

Journal Publication

Version 1

Abstract

Figures

Introduction

Results and Discussion

Norleucine-ε-dimethylsulfonium as a methyllysine mimic

Mechanism for photo crosslinking of NleSme2

NleSme2-tryptophan crosslinking is site-selective

Investigating nuclear reader proteins using NleS+me2 probes

Applicability of sulfonium-tryptophan crosslinking

Conclusions

Methods

Declarations

Competing interests

Author contributions

Acknowledgments

Data availability

Reporting summary

References

Additional Declarations

Supplementary Files

Status:

Journal Publication

Version 1

Investigating nuclear reader proteins using NleS⁺me2 probes