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Pseudokinases can catalyse peptide cyclization through thioether crosslinking

Nature Chemistry (2025)Cite this article

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Abstract

The protein-kinase-like superfamily proteins are crucial and generally catalyse substrate phosphorylation using adenosine 5′-triphosphate. Pseudokinases are non-canonical protein-kinase-like members deficient in kinase activity, and few of them are known to be enzymatically active and to have catalytic ability rather than phosphorylation. Based on biosynthetic investigations into thioamitides and lanthipeptides—two different families of ribosomally synthesized and post-translationally modified peptides—we here report a peptide cyclization activity of pseudokinases (TvaE and SacE) that enables (ene)thioether residue formation. We determine the dedicated cyclase activity in unsaturated 2-aminovinyl-cysteine formation and mine for similar activity in saturated lanthionine formation. Biochemical characterization, heterologous expression, co-crystallization, computational analysis, genome mining, isotope labelling and site-specific mutagenesis rationalize the commonality in catalysis, demonstrating that a protein-kinase fold can be repurposed for unexpected utilities. Related cyclases differ from known enzymatically active pseudokinases that resemble canonical protein-kinase-like proteins in mechanism and function. Instead, they catalyse Michael addition for (ene)thioether crosslinking through a sandwich-like substrate-assisted process.

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Fig. 1: Thioamitide-related structure, biosynthetic genes and enzymatic process.
Fig. 2: Characterization of the individual roles of TvaE and TvaF in AviCys formation.
Fig. 3: Characterization of the interactions between TvaE and the LP sequence of TvaA.
Fig. 4: Mechanistic analysis of AviCys formation in TvaE.
Fig. 5: Characterization of the sac cluster.
Fig. 6: Mechanistic analysis of Lan formation in SacE.

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Data availability

The data underlying the findings of this study are available in this Article and its Supplementary Information. The atomic coordinates of TvaE have been deposited in the PDB (http://www.rcsb.org) with the accession number 8JSU. Source data are provided with this paper.

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Acknowledgements

We thank C. Buhlheller from the Medical University of Graz, Austria, for help in solving the phase problem of the TvaE–LP1–LP52 complex. This work is part of the Shanghai Municipal Science and Technology Major Project and was supported in part by grants from the National Natural Science Foundation of China (22193070, 32030002 and 22537007), the Shanghai Commission of Science and Technology (24HC2820300), the Strategic Priority Research Program of the Chinese Academy of Sciences (grant no. XDB1060000) and the CAS Youth Interdisciplinary Team (JCTD-2022-10). The funders had no role in study design, data collection and analysis, and decision to publish or preparation of the manuscript.

Author information

Author notes

  1. These authors contributed equally: Ling Hu, Miao Li, Yueqian Sang, Chengxin Zhao.

Authors and Affiliations

  1. School of Life Science & Biotechnology, Shanghai Jiao Tong University, Shanghai, China

    Ling Hu, Yanqing Xue & Wen Liu

  2. State Key Laboratory of Chemical Biology, Shanghai Institute of Organic Chemistry, University of Chinese Academy of Sciences, Chinese Academy of Sciences, Shanghai, China

    Ling Hu, Miao Li, Jiwu Huang, Wei Huang, Yanqing Xue, Lei Liu, Lifeng Pan & Wen Liu

  3. Zhangjiang Institute for Advanced Study, Shanghai Jiao Tong University, Shanghai, China

    Ling Hu & Wen Liu

  4. School of Chemistry and Materials Science, Hangzhou Institute for Advanced Study, University of Chinese Academy of Sciences, Hangzhou, China

    Miao Li & Lifeng Pan

  5. State Key Laboratory of Fluorine and Nitrogen Chemistry and Advanced Materials, Shanghai Institute of Organic Chemistry, University of Chinese Academy of Sciences, Chinese Academy of Sciences, Shanghai, China

    Yueqian Sang & Xiao-Song Xue

  6. Department of Chemistry, Xi’an Jiaotong University, Xi’an, China

    Chengxin Zhao & Pengchen Ma

  7. CAS Key Laboratory of Photochemistry, Institute of Chemistry, Chinese Academy of Sciences, Beijing, China

    Chengxin Zhao

  8. Syngenta Jealott’s Hill International Research Centre, Bracknell, United Kingdom

    Yucheng Gu

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Contributions

L.H., W.H. and Y.X. performed in vivo and in vitro experiments. L.H. and J.H. conducted chemical characterization. M.L., L.L. and L.P. carried out protein crystallization and structure determination. Y.S., C.Z., P.M. and X.-S.X. performed computational studies. L.H., M.L., Y.S., C.Z., J.H., W.H., Y.X., L.L., Y.G., P.M., X.-S.X., L.P. and W.L. analysed data and discussed results. L.H., P.M., X.-S.X., L.P. and W.L. prepared the paper. W.L. designed and organized the research.

Corresponding authors

Correspondence to Pengchen Ma, Xiao-Song Xue, Lifeng Pan or Wen Liu.

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Nature Chemistry thanks Yousong Ding, Yong-Xin Li, Satish Nair and the other anonymous reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Schematic representations of reactions catalyzed by PKL proteins.

(a) Canonical protein kinase-catalyzed phosphorylation; (b) SelO-catalyzed AMPylation; (c) SidJ-catalyzed glutamylation; (d) nsp12-catalyzed RNAylation.

Extended Data Fig. 2 Examination of the LP dependence of TvaE activity.

(a) Structures of 1, 1e and derivatives. The cleavage site of trypsin is indicated by dashed line. (b) Conversion of 1e. Following the treatment of 1 with trypsin, the conversions of resultant 1e were conducted in the absence of any enzymes (i) and in the presence of TvaE (ii), TvaF (iii) and TvaEF (iv), respectively. For HR-MS analysis of 1e, 3e, 3f and 3g, the dashed lines indicate the monoisotopic peaks of ESI m/z [M + 3H]3+ (calculated 706.0209, 751.6857, 732.3566 and 746.3601, respectively. For observed details and errors, see Supplementary Figs. 13, 15, 18 and 19). Related experiments were performed >= 3 times (with >= 2 parallel samples for each).

Extended Data Fig. 3 Determination of the necessity of selected residues for interactions between TvaE and the LP sequence of TvaA.

(a) Examination of the binding affinities by ITC. TvaE or its variant was titrated with LP1–52 or its variant. (b) Conversions of 1 derivatives. Samples including 1I21R (i) and the mixtures of 1I21R with TvaE (ii), TvaF (iii) or TvaEF (iv), and 1E25R (v) and the mixtures of 1E25R with TvaE (vi), TvaF (vii) or TvaEF (viii), respectively, were examined after incubation 30 °C for 2-h. For HR-MS analysis of 1I21R, 3I21R, 1E25R, and 3E25R, the dashed lines indicate the monoisotopic peaks of ESI m/z [M + 7H]7+ (calculated 1289.1874, 1282.6212, 1286.9076, and 1280.3361, respectively. For observed details and errors, see Supplementary Figs. 25–28). The mutation sites in the precursor peptide are indicated by asterisks. (c) Conversions of 1. Samples including the mixtures of 1 with TvaF (i), TvaEF (ii), TvaEVal23GlnF (iii) and TvaEArg32GluF (iv), respectively, were examined after incubation 30 °C for 2-h. For HR-MS analysis of 1, 2 or 3, 3a and 3b (left), the dashed lines indicate the monoisotopic peaks of ESI m/z [M + 6H]6+; and for EICs in HPLC-HR-MS analysis (right), the ESI m/z [M + 6H]6+ mode for both 2 and 3 is 1489.88. (d) Analysis of the production of thioamitide in S. coelicolor. For EIC in HPLC-HR-MS analysis, the ESI m/z [M + H]+ mode for TVA-YJ-2 is 1305.5. Tested strains include the S. coelicolor strains harboring a wild-type tva gene cluster (i) and an engineered cluster to encode mutant TvaAI21R (ii), TvaAE25R (iii), TvaEVal23Gln (iv) or TvaEArg32Glu (v), respectively. HPLC peaks corresponding to the different products are highlighted by color: green for 2, and light blue for TVA-YJ-2. Related experiments were performed >= 3 times (with >= 2 parallel samples for each).

Source data

Extended Data Fig. 4 Investigations into the catalytically necessary residues in TvaE.

(a) Examination of the binding affinities by ITC. TvaE variant was titrated with LP1–52, and obtained KD values are shown. (b) Conversions of 1. Samples including the mixtures of 1 with TvaF (i), TvaEF (ii), TvaEArg220AlaF (iii), TvaEGln224AlaF (iv) and TvaEHis273AlaF (v), respectively, were examined after incubation 30 °C for 2-h. For HR-MS analysis of 1, 2 or 3, 3a and 3b (left), the dashed lines indicate the monoisotopic peaks of ESI m/z [M + 6H]6+; and for EICs in HPLC-HR-MS analysis (right), the ESI m/z [M + 6H]6+ mode for both 2 and 3 is 1489.88. (c) Analysis of the production of thioamitide in S. coelicolor. For EIC in HPLC-HR-MS analysis, the ESI m/z [M + H]+ mode for TVA-YJ-2 is 1305.5. Tested strains include the S. coelicolor strains harboring a wild-type tva gene cluster (i) and an engineered cluster to encode mutant TvaEArg220Ala (ii), TvaEGln224Ala (iii), or TvaEHis273Ala (iv), respectively. HPLC peaks corresponding to the different products are highlighted by color: green for 2, and light blue for TVA-YJ-2. Related experiments were performed >= 3 times (with >= 2 parallel samples for each).

Source data

Extended Data Fig. 5 QM/MM-calculated energy profile (kcal/mol) for the formation of AviCys in TvaE, along with schematic drawings of the reactant, key intermediates and product.

3D structures of intermediate and transition states are shown in bottom. Key distances are given in Å. At the beginning, the Nδ1 atom of the imidazole ring in H86 abstracts a proton from the enethiol group via transition state TS1 (with a low energy barrier of 6.3 kcal/mol), leading to intermediate IM1 with 3.2 kcal/mol energy. Notably, H86 functions as a general base39. This finding aligns with the experiment result from H86 mutation studies. Subsequently, the protonated imidazole group involves a ring flipping to form intermediate IM2 with 7.1 kcal/mol energy40,41, facilitating the following Michael addition39,42. Finally, with the assistant of His273 in TvaE, Michael addition occurs by overcoming an energy barrier of 17.7 kcal/mol (TS2), yields the exergonic AviCys-containing product by −12.8 kcal/mol energy43,44. Overall, the reaction proceeds via a stepwise pathway, with the two imidazole groups of H86 and H273 functioning as a general base and a proton donor, respectively. His273 is further stabilized by Gln224.

Extended Data Fig. 6 Investigations into the catalytic function of H86 in CP.

(a) Conversions of 1 variants. Samples including 1H86A (i) and the mixtures of 1H86A with TvaE (ii), TvaF (iii) or TvaEF (iv), and 1H86F (v) and the mixtures of 1H86F with TvaE (vi), TvaF (vii) or TvaEF (viii), respectively, were examined after incubation 30 °C for 2-h. For HR-MS analysis of 1H86A, 3bH86A, 1H86F, and 3bH86F, the dashed lines indicate the monoisotopic peaks of ESI m/z [M + 6H]6+ (calculated 1485.7110, 1475.3814, 1498.3829, and 1488.0533, respectively. For observed details and errors, see Supplementary Figs. 36–39). The mutation sites in the precursor peptide are indicated by asterisks. (b) Analysis of the production of thioamitides in S. coelicolor. For EIC in HPLC-HR-MS analysis, the ESI m/z [M + H]+ mode for TVA-YJ-2, TVA-YJ-2H86A, and TVA-YJ-2H86F are 1305.5. 1195.5, and 1271.5, respectively. Tested strains include the S. coelicolor strains harboring a wild-type tva gene cluster (i) and an engineered cluster to encode mutant TvaAH86A (ii), or TvaAH86F (iii), respectively. The HPLC peak corresponding to the TVA-YJ-2 variants is highlighted by light blue. Related experiments were performed >= 3 times (with >= 2 parallel samples for each).

Extended Data Fig. 7 SacE-catalyzed Lan thioether opening and recyclization.

Proposed mechanisms for α-proton exchange in D2O (path A) and NEM derivatization (path B) were shown.

Extended Data Fig. 8 Investigations into the substrate selectivity of SacE.

(a) Analysis of proton exchange through enzymatic ring opening and recylization in D2O. (i), treatment of 4 (top, control) with SacE (bottom); and (ii), treatment of 5 (top, control) with SacE (bottom). (b) Analysis of 7 production based on NEM derivatization. (i), treatment of 4 (top, control) with SacE (bottom); and (ii), treatment of 5 (top, control) with SacE (bottom) in the presence of NEM, respectively. In panels (a) and (b), for HR-MS analysis of 4 and 5, the dashed lines indicate the monoisotopic peaks of ESI m/z [M + 4H]4+ (calculated 1426.2039. See Supplementary Figs. 43 for observed details and errors). Related experiments were performed >= 3 times (with >= 2 parallel samples for each).

Extended Data Fig. 9 Analysis of 7 variants production based on NEM derivatization.

Related experiments were performed >= 3 times (with >= 2 parallel samples for each). (i), treatment of 6 with NEM (top), 6 with SacE (middle) or 6 with SacE and NEM (bottom); (ii), treatment of 6 with NEM (top), 6 with SacEArg261Ala (middle) or 6 with SacEArg261Ala and NEM (bottom); and (iii), treatment of 6D19A with NEM (top), 6D19A with SacE (middle) or 6D19A with SacE and NEM (bottom). For HR-MS analysis of 6, 7-NEM, 6D19A or 7D19A, and 7D19A -NEM, the dashed lines indicate the monoisotopic peaks of ESI m/z [M + 4H]4+ (calculated 1426.2039, 1457.4658, 1415.2064 and 1446.4683, respectively. See Supplementary Figs. 44, 52, 55, 57 and 58 for observed details and errors).

Extended Data Fig. 10 Determination of the necessity of polar residues in the precursor peptide for Lan formation.

(a) Precursor peptide SacA. The mutation sites in the precursor peptide are indicated by asterisks. (b) Analysis of deuterium exchange through enzymatic ring opening and recylization in D2O. (i), treatment of 6E30A (top, control) with SacE (bottom); (ii), treatment of 6E33A (top, control) with SacE (bottom); and (iii), treatment of 6D53A (top, control) with SacE (bottom) in the mixture containing 50 mM Tris-HCl (pH 7.5). (c) Analysis of 7 variant production based on NEM derivatization. (i), treatment of 6E30A (top, control) with SacE (bottom); (ii), treatment of 6E33A (top, control) with SacE (bottom); and (iii), treatment of 6D53A (top, control) with SacE (bottom) in the presence of NEM. (d) Analysis of deuterium exchange of 6D19A under basic conditions. Samples include 6D19A (top, control) and the mixture of 6D19A (top, control) with SacE (bottom) and 50 mM Tris-HCl (pH 9.0) in D2O. (e) Proposed mechanism of D19-involved thiol deprotonation during retro-Michael and Michael reactions. For panels b, c and d, related experiments were performed >= 3 times (with >= 2 parallel samples for each).

Supplementary information

Supplementary Information

Supplementary Notes 1 and 2, Figs. 1–71, Tables 1–5, Methods, references and uncropped scans of gels.

Reporting Summary

Supplementary Data 1

The xyz coordinates.

Supplementary Data 2

Protein and DNA sequences, per Supplementary Section 1.4.

Source data

Source Data Fig. 3

Statistical source data.

Source Data Extended Data Fig. 3

Statistical source data.

Source Data Extended Data Fig. 4

Statistical source data.

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Hu, L., Li, M., Sang, Y. et al. Pseudokinases can catalyse peptide cyclization through thioether crosslinking. Nat. Chem. (2025). https://doi.org/10.1038/s41557-025-01954-1

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