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Dithiol amino acids can structurally shape and enhance the ligand-binding properties of polypeptides

Nature Chemistry volume 6, pages 1009–1016 (2014)Cite this article

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Abstract

The disulfide bonds that form between two cysteine residues are important in defining and rigidifying the structures of proteins and peptides. In polypeptides containing multiple cysteine residues, disulfide isomerization can lead to multiple products with different biological activities. Here, we describe the development of a dithiol amino acid (Dtaa) that can form two disulfide bridges at a single amino acid site. Application of Dtaas to a serine protease inhibitor and a nicotinic acetylcholine receptor inhibitor that contain disulfide constraints enhanced their inhibitory activities 40- and 7.6-fold, respectively. X-ray crystallographic and NMR structure analysis show that the peptide ligands containing Dtaas have retained their native tertiary structures. We furthermore show that replacement of two cysteines by Dtaas can avoid the formation of disulfide bond isomers. With these properties, Dtaas are likely to have broad application in the rational design or directed evolution of peptides and proteins with high activity and stability.

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Figure 1: Constraining peptide conformation and reducing the number of disulfide bond isomers in disulfide-rich polypeptides by applying Dtaas.
Figure 2: Substituting adjacent cysteines with Dtaas in a protease inhibitor.
Figure 3: X-ray structures of bicyclic peptides bound to the active site of uPA.
Figure 4: α-conotoxin ImI and Dtaa derivatives thereof.
Figure 5: Dtaas with symmetrically branched side chains.

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Acknowledgements

The authors thank the staff of beamlines PXIII and PXI of the Swiss Light Source at the Paul Scherrer Institute (SLS, Villigen, Switzerland) for providing technical assistance during data collection. The authors also thank X. Hu for help with the ultraviolet source for the radical reaction and M. Bogyo for discussions and critical reading of the manuscript. The authors acknowledge support from the NCCR Chemical Biology. Financial contributions from the Swiss National Science Foundation (SNSF Professorship PP00P3_123524/1 to C.H. and research grant 31003-13314 to R.H.) and the EPFL are acknowledged.

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Authors and Affiliations

  1. Institute of Chemical Sciences and Engineering, Ecole Polytechnique Fédérale de Lausanne, Lausanne, CH-1015, Switzerland

    Shiyu Chen, Ranganath Gopalakrishnan, Ruud Hovius & Christian Heinis

  2. HiQScreen, Vésenaz, CH-1022, Geneva, Switzerland

    Tifany Schaer, Fabrice Marger & Daniel Bertrand

  3. Protein Crystallography Core Facility, School of Life Sciences, Ecole Polytechnique Fédérale de Lausanne, Lausanne, CH-1015, Switzerland

    Florence Pojer

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  1. Shiyu Chen
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  2. Ranganath Gopalakrishnan
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  8. Christian Heinis
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Contributions

C.H. and S.C. conceived the project and designed the experiments. S.C. and R.G. synthesized the amino acids and the peptides. S.C. and F.P. solved the X-ray structures. S.C. solved the NMR structure. D.B. and R.H. planned and analysed the electrophysiology experiments, which were performed by T.S. and F.M. C.H. and S.C. analysed the data and wrote the manuscript with input from all other authors.

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Correspondence to Christian Heinis.

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The authors declare no competing financial interests.

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Chen, S., Gopalakrishnan, R., Schaer, T. et al. Dithiol amino acids can structurally shape and enhance the ligand-binding properties of polypeptides. Nature Chem 6, 1009–1016 (2014). https://doi.org/10.1038/nchem.2043

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