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Tyrosine sulfation modulates activity of tick-derived thrombin inhibitors

Nature Chemistry volume 9, pages 909–917 (2017)Cite this article

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Abstract

Madanin-1 and chimadanin are two small cysteine-free thrombin inhibitors that facilitate blood feeding in the tick Haemaphysalis longicornis. Here, we report a post-translational modification—tyrosine sulfation—of these two proteins that is critical for potent anti-thrombotic and anticoagulant activity. Inhibitors produced in baculovirus-infected insect cells displayed heterogeneous sulfation of two tyrosine residues within each of the proteins. One-pot ligation–desulfurization chemistry enabled access to homogeneous samples of all possible sulfated variants of the proteins. Tyrosine sulfation of madanin-1 and chimadanin proved crucial for thrombin inhibitory activity, with the doubly sulfated variants three orders of magnitude more potent than the unmodified inhibitors. The three-dimensional structure of madanin-1 in complex with thrombin revealed a unique mode of inhibition, with the sulfated tyrosine residues binding to the basic exosite II of the protease. The importance of tyrosine sulfation within this family of thrombin inhibitors, together with their unique binding mode, paves the way for the development of anti-thrombotic drug leads based on these privileged scaffolds.

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Figure 1: Madanin-1 and chimadanin: thrombin inhibitors from the bush tick Haemaphysalis longicornis.
Figure 2: Assembly of homogeneously sulfated madanin-1 variants through one-pot kinetically controlled ligation-desulfurization in the N- to C-direction.
Figure 3: Assembly of differentially sulfated chimadanin proteins through one-pot sequential native chemical ligation reactions, followed by desulfurization.
Figure 4: Tyrosine sulfation strongly modulates the anti-thrombin activity of madanin-1 and chimadanin.
Figure 5: Madanin-1 blocks both the active site and the exosite II regions of human α-thrombin.
Figure 6: Madanin-1 and chimadanin belong to a new mechanistic family of exosite II binding inhibitors.

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Acknowledgements

The research was supported by an Australian Research Council Future Fellowship (to R.J.P.), by Australian Postgraduate Awards (to R.E.T. and X.L.) and a John Lamberton Research Scholarship (to R.E.T.). This work was funded in part by the European Social Fund through Programa Operacional Capital Humano (POCH) and by national funds through Fundação para a Ciência e a Tecnologia (Portugal) in the form of a postdoctoral fellowship SFRH/BPD/108004/2015 (to J.R.-R.). We acknowledge the ESRF for provision of synchrotron radiation facilities and thank the ESRF staff for help with data collection. Transnational Access to the High Throughput Crystallization Laboratory of the European Molecular Biology Laboratory Grenoble Outstation was supported by the European Community-Seventh Framework Program (FP7/2007-2013) Grant Protein Production Platform (PCUBE agreement no. 227764).

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  1. Robert E. Thompson, Xuyu Liu and Jorge Ripoll-Rozada: These authors contributed equally to this work

Authors and Affiliations

  1. School of Chemistry, The University of Sydney, Sydney, New South Wales 2006, Australia

    Robert E. Thompson, Xuyu Liu & Richard J. Payne

  2. IBMC – Instituto de Biologia Molecular e Celular, Universidade do Porto, Porto, 4200-135, Portugal

    Jorge Ripoll-Rozada, Noelia Alonso-García & Pedro José Barbosa Pereira

  3. Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Porto, 4200-135, Portugal

    Jorge Ripoll-Rozada, Noelia Alonso-García & Pedro José Barbosa Pereira

  4. Charles Perkins Centre, The University of Sydney, 2006, New South Wales, Australia.

    Benjamin L. Parker

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Contributions

R.E.T., X.L. and J.R.-R. contributed equally to this work. R.E.T. and X.L. synthesized the thiol aspartate and glutamate amino acids, developed and employed the ligation methods for the assembly of differentially sulfated madanin-1 and chimadanin peptides and proteins and performed all the characterization experiments; J.R.-R. carried out all baculovirus expression experiments, some of the thrombin inhibition experiments with the synthetic madanin-1 and chimadanin sulfoproteins and determined the structure of the thrombin-madanin-1 complex; N.A.-G. performed some of the thrombin inhibition experiments with the synthetic madanin-1 and chimadanin sulfoproteins; B.L.P. performed the liquid chromatography–tandem mass spectrometry experiments on madanin-1 and chimadanin expressed in baculovirus; P.J.B.P. and R.J.P designed and directed the investigations and composed the manuscript together with the other authors.

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Correspondence to Richard J. Payne.

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Thompson, R., Liu, X., Ripoll-Rozada, J. et al. Tyrosine sulfation modulates activity of tick-derived thrombin inhibitors. Nature Chem 9, 909–917 (2017). https://doi.org/10.1038/nchem.2744

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