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Crystal structure of the MAPK phosphatase Pyst1 catalytic domain and implications for regulated activation

Nature Structural Biology volume 6, pages 174–181 (1999)Cite this article

Abstract

The crystal structure of the catalytic domain from the MAPK phosphatase Pyst1 (Pyst1–CD) has been determined at 2.35 Å. The structure adopts a protein tyrosine phosphatase (PTPase) fold with a shallow active site that displays a distorted geometry in the absence of its substrate with some similarity to the dual–specificity phosphatase cdc25. Functional characterization of Pyst1–CD indicates it is sufficient to dephosphorylate activated ERK2 in vitro. Kinetic analysis of Pyst1 and Pyst1–CD using the substrate p–nitrophenyl phosphate (pNPP) reveals that both molecules undergo catalytic activation in the presence of recombinant inactive ERK2, switching from a low– to high–activity form. Mutation of Asp 262, located 5.5 Å distal to the active site, demonstrates it is essential for catalysis in the high–activity ERK2–dependent conformation of Pyst1 but not for the low–activity ERK2–independent form, suggesting that ERK2 induces closure of the Asp 262 loop over the active site, thereby enhancing Pyst1 catalytic efficiency.

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Figure 1: a, Overall fold for Pyst1–CD.
Figure 2: Sequence alignment for MKPs and secondary structure elements of Pyst1–CD (top) and VHR (bottom).
Figure 3: a, Stereoview of the 2Fo – Fc electron density for sequences close to the PTP–loop of the Pyst1 C293S mutant, contoured at 1.0σ
Figure 4: Electrostatic surface of Pyst1–CD drawn using GRASP30.
Figure 5: Inactivation and dephosphorylation of 32P–labeled ERK2 by Pyst1–CD.
Figure 6: A hypothetical model for the interaction of Pyst1 with ERK2.

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References

  1. Keyse, S.M. Protein phosphatase and the regulation of MAP kinase activity. Semin. Cell. Dev. Biol. 9, 143–152 (1998).

    Article  CAS  Google Scholar 

  2. Cobb, M & Goldsmith E.J. How MAP kinases are regulated J. Biol. Chem. 270, 14843– 14846 (1995).

    Article  CAS  Google Scholar 

  3. Fauman, E.B., & Saper, M.A. Structure and function of the protein tyrosine phosphatases. Trends Biochem. Sci. 21, 413–417 (1996).

    Article  CAS  Google Scholar 

  4. Keyse, S.M. & Ginsburg, M. Amino acid sequence similarity between CL100, a dual–specificity MAP kinase phosphatase and cdc25. Trends Biochem. Sci. 18, 377– 378 (1993).

    Article  CAS  Google Scholar 

  5. Fauman, E.B. et al. Crystal structure of the catalytic domain of the human cell cycle control phosphatase, CDC25A. Cell. 93, 617–625 (1998).

    Article  CAS  Google Scholar 

  6. Keyse, S.M. & Emslie, E.A. Oxidative stress and heat shock induce a human gene encoding a protein–tyrosine phosphatase. Nature 359, 644–647 ( 1992).

    Article  CAS  Google Scholar 

  7. Treisman, R. Regulation of transcription by MAP kinase cascades. Curr. Opin. Cell. Biol. 8, 205–215 ( 1996).

    Article  CAS  Google Scholar 

  8. Groom, L. A., Sneddon, A. A., Alessi, D. R., Dowd, S. & Keyse, S.M. Differential regulation of the MAP, SAP and RK/p38 kinases by Pyst1, a novel cytosolic dual–specificity phosphatase. EMBO J. 15, 3621– 3632 (1996).

    Article  CAS  Google Scholar 

  9. Muda, M. et al. The mitogen–activated protein kinase phosphatase–3 N–terminal noncatalytic domain is responsible for tight substrate binding and enzymatic specificity. J. Biol.Chem. 273, 9323– 9329 (1998).

    Article  CAS  Google Scholar 

  10. Camps, M. et al. Catalytic activation of the phosphatase MKP–3 by ERK2 mitogen–activated protein kinase. Science 280, 1262– 1265 (1998).

    Article  CAS  Google Scholar 

  11. Yuvaniyama,J., Denu, J.M., Dixon, J.E. & Saper, M.A. Crystal structure of the dual–specificity protein phosphatase VHR. Science 272, 1328–1331 ( 1996).

    Article  CAS  Google Scholar 

  12. Barford, D., Flint, A.J. & Tonks, N.K. Crystal structure of human protein tyrosine phosphatase 1B. Science 263, 1397–1404 (1994).

    Article  CAS  Google Scholar 

  13. Stuckey, J.A., Schubert, H.L., Fauman, E.B., Zhang, Z.Y., Dixon, J.E. & Saper, M.A. Crystal structure of Yersinia protein tyrosine phosphatase at 2.5 Å and the complex with tungstate. Nature 370, 571–575 (1994).

    Article  CAS  Google Scholar 

  14. Schneider,G., Lindqvist, Y. & Vihko, P. Three–dimensional structure of rat acid phosphatase. EMBO J. 12, 2609–2615 (1993).

    Article  CAS  Google Scholar 

  15. Canagarajah, B.J., Khokhlatchev,A., Cobb, M.H. & Goldsmith, E.J. Activation mechanism of the MAP kinase ERK2 by dual phosphorylation. Cell 90, 859–869 ( 1997).

    Article  CAS  Google Scholar 

  16. Denu, Je, D.L., Vijayalakshmi, J., Saper, M.A. & Dixon, J.E. Visualization of intermediate and transition–state structures in protein–tyrosine phosphatase catalysis. Proc. Natl. Acad. Sci. USA 93 , 2493–2498 (1996).

    Article  Google Scholar 

  17. Flint, A.J., Tiganis, T., Barford, D. & Tonks, N.K. Development of "substrate–trapping" mutants to identify physiological substrates of protein tyrosine phosphatases. Proc. Natl. Acad. Sci. USA 94, 1680–1685 (1997).

    Article  CAS  Google Scholar 

  18. Wiland, A.M., Denu, J.M., Mourey, R.J. & Dixon, J.E. Purification and kinetic characterization of the mitogen–activated protein kinase phosphatase rVH6. J. Biol. Chem., 271, 33486– 33492 (1996).

    Article  CAS  Google Scholar 

  19. Jia, Z., Barford, D., Flint, A.J & Tonks, N.K. Structural basis for phosphotyrosine peptide recognition by protein tyrosine phosphatase 1B Science 268, 1754–1758 (1995).

    Article  CAS  Google Scholar 

  20. Denu, J.M., Zhou, G., Guo, Y. & Dixon, J.E. The catalytic role of aspartic acid–92 in a human dual–specific protein–tyrosine phosphatase. Biochemistry 34, 3396– 3403 (1995).

    Article  CAS  Google Scholar 

  21. Ausebel, F.M. et al. Current protocols in molecular biology. (John Wiley & Sons, Inc., New York; 1987).

    Google Scholar 

  22. Hendrickson, W.A., Horton, J.R. & Lemaster, D.M. Selenomethionyl proteins produced for analysis by multiwavelength anomalous diffraction (MAD)—a vehicle for direct determination of 3–dimensional structure. EMBO J. 9, 1665–1672 (1990).

    Article  CAS  Google Scholar 

  23. Zhang, Z.Y., Maclean, D., Thiemesefler, A.M., Roeske, R.W. & Dixon, J.E. A continuous spectrophotometric and fluorimetric assay for protein tyrosine phosphatase using phospho–tyrosine–containing peptides. Anal. Biochem. 211, 7– 15 (1993).

    Article  CAS  Google Scholar 

  24. Otwinowski, Z. Oscillation data reduction program, in Proceedings of the CCP4 study weekend: Data collection and processing, 29–30 January (compilers Sawyer, L., Issacs, N & Bailey, S.) 55–62 (SERC Daresbury Laboratory, UK; 1993).

    Google Scholar 

  25. Collaborative Computational Project, Number 4 The CCP4 suite: Programs for protein crystallography. Acta Crystallogr. D 50, 760–763 ( 1994).

  26. Brünger, A.T. X–PLOR version 3.1: A system for X–ray crystallography and NMR (Yale University, Department of Molecular Biophysics, New Haven, CT; 1992).

    Google Scholar 

  27. Hendrickson, W.A. Transformation to optimize the superposition of similar structures. Acta. Crystallogr. A35, 158–163 (1979).

    Article  CAS  Google Scholar 

  28. Jones, T.A., Zou, J.Y., Cowan, S.W. & Kjeldgaard, M. Improved methods for building protein models in electron density maps and the location of errors in these models. Acta Crystallogr. A47, 110–119 (1991).

    Article  CAS  Google Scholar 

  29. Laskowski, R.A., Macarthur, M.W., Moss, D.S. & Thornton, J.M. PROCHECK—a program to check the stereochemical quality of protein structures. J. Appl. Crystallogr. 26, 283– 291 (1993).

    Article  CAS  Google Scholar 

  30. Nicholls, A., Sharp, K. & Honig, B. Protein folding and association: Insights from the interfacial and thermodynamic properties of hydrocarbons. Proteins 11, 281–296 (1991).

    Article  CAS  Google Scholar 

  31. Kraulis, P.J. MOLSCRIPT: A program to produce both detailed and schematic plots of protein structures. J. Appl. Crystallogr. 24, 946 –950 (1991).

    Article  Google Scholar 

  32. Evans, S.V. SETOR: Hardware lighted three–dimensional solid model representations of macromolecules. J. Mol. Graphics 11, 134–138 (1993).

    Article  CAS  Google Scholar 

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Acknowledgements

We are grateful to D. Alessi for assistance in the phospho–amino acid analysis of 32P–labeled ERK2. A.E.S. would like to thank all members of the Structural Biology Laboratory for helpful discussions and encouragement. We also thank J. Murray–Rust for helpful comments on the manuscript and gratefully acknowledge the use of SRS facilities at Daresbury.

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Authors and Affiliations

  1. Structural Biology Laboratory, Imperial Cancer Research Fund, 44 Lincolns Inn Fields, London, WC2A 3PX, UK

    Albert E. Stewart & Neil Q. McDonald

  2. ICRF Molecular Pharmacology Unit, Biomedical Research Centre, Ninewells Hospital, Dundee, DD1 9SY, UK

    Stephen Dowd & Stephen M. Keyse

  3. Department of Crystallography, Birkbeck College, Malet St., London, WC1E 7HX, UK

    Neil Q. McDonald

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Correspondence to Neil Q. McDonald.

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Stewart, A., Dowd, S., Keyse, S. et al. Crystal structure of the MAPK phosphatase Pyst1 catalytic domain and implications for regulated activation. Nat Struct Mol Biol 6, 174–181 (1999). https://doi.org/10.1038/5861

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