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Structure Determination of SH2–Phosphopeptide Complexes by X-Ray Crystallography: The Example of p120RasGAP

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SH2 Domains

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2705))

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Abstract

The p120RasGAP protein contains two Src homology 2 (SH2) domains, each with phosphotyrosine-binding activity. We describe the crystallization of the isolated and purified p120RasGAP SH2 domains with phosphopeptides derived from a binding partner protein, p190RhoGAP. Purified recombinant SH2 domain protein is mixed with synthetic phosphopeptide at a stoichiometric ratio to form the complex in vitro. Crystallization is then achieved by the hanging drop vapor diffusion method over specific reservoir solutions that yield single macromolecular co-crystals containing SH2 domain protein and phosphopeptide. This protocol yields suitable crystals for X-ray diffraction studies, and our recent X-ray crystallography studies of the two SH2 domains of p120RasGAP demonstrate that the N-terminal SH2 domain binds phosphopeptide in a canonical interaction. In contrast, the C-terminal SH2 domain binds phosphopeptide via a unique atypical binding mode. The crystallographic studies for p120RasGAP illustrate that although the three-dimensional structure of SH2 domains and the molecular details of their binding to phosphotyrosine peptides are well defined, careful structural analysis can continue to yield new molecular-level insights.

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Acknowledgments

We acknowledge Rachel Jaber Chehayeb and Jessica Wang. Kimberly Vish is acknowledged for helpful comments. This research was supported by 1R01NS117609 and 1R01GM138411 to T.J.B.

Author information

Authors and Affiliations

  1. Department of Pharmacology, Yale University, New Haven, CT, USA

    Amy L. Stiegler & Titus J. Boggon

  2. Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT, USA

    Titus J. Boggon

  3. Yale Cancer Center, Yale University, New Haven, CT, USA

    Titus J. Boggon

Authors
  1. Amy L. Stiegler
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  2. Titus J. Boggon
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Correspondence to Titus J. Boggon .

Editor information

Editors and Affiliations

  1. School of Biosciences, College of Life and Environmental Sciences, University of Birmingham, Birmingham, UK

    Teresa Carlomagno

  2. Signalling Research Centres BIOSS &CIBSS, University of Freiburg, Freiburg, Germany

    Maja Köhn

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© 2023 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature

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Stiegler, A.L., Boggon, T.J. (2023). Structure Determination of SH2–Phosphopeptide Complexes by X-Ray Crystallography: The Example of p120RasGAP. In: Carlomagno, T., Köhn, M. (eds) SH2 Domains. Methods in Molecular Biology, vol 2705. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3393-9_5

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  • DOI: https://doi.org/10.1007/978-1-0716-3393-9_5

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-3392-2

  • Online ISBN: 978-1-0716-3393-9

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