Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Technical Report
  • Published:

A potent and highly specific FN3 monobody inhibitor of the Abl SH2 domain

Nature Structural & Molecular Biology volume 17, pages 519–527 (2010)Cite this article

Subjects

Abstract

Interactions between Src homology 2 (SH2) domains and phosphotyrosine sites regulate tyrosine kinase signaling networks. Selective perturbation of these interactions is challenging due to the high homology among the 120 human SH2 domains. Using an improved phage-display selection system, we generated a small antibody mimic (or 'monobody'), termed HA4, that bound to the Abelson (Abl) kinase SH2 domain with low nanomolar affinity. SH2 protein microarray analysis and MS of intracellular HA4 interactors showed HA4's specificity, and a crystal structure revealed how this specificity is achieved. HA4 disrupted intramolecular interactions of Abl involving the SH2 domain and potently activated the kinase in vitro. Within cells, HA4 inhibited processive phosphorylation activity of Abl and also inhibited STAT5 activation. This work provides a design guideline for highly specific and potent inhibitors of a protein interaction domain and shows their utility in mechanistic and cellular investigations.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Library design and selected Abl SH2–binding monobodies.
Figure 2: SH2 protein microarray assays of HA4 specificity.
Figure 3: The crystal structure of the HA4–Abl SH2 complex.
Figure 4: Structural basis for HA4′s specificity toward Abl and Abl2.
Figure 5: HA4 activates autoinhibited Abl.
Figure 6: HA4 blocks processive phosphorylation of an Abl substrate in cells and inhibits STAT5 phosphorylation in leukemia cells.

Similar content being viewed by others

Accession codes

Primary accessions

Protein Data Bank

References

  1. Pawson, T. & Nash, P. Assembly of cell regulatory systems through protein interaction domains. Science 300, 445–452 (2003).

    Article  CAS  Google Scholar 

  2. Rual, J.F. et al. Towards a proteome-scale map of the human protein-protein interaction network. Nature 437, 1173–1178 (2005).

    Article  CAS  Google Scholar 

  3. Kocher, T. & Superti-Furga, G. Mass spectrometry-based functional proteomics: from molecular machines to protein networks. Nat. Methods 4, 807–815 (2007).

    Article  Google Scholar 

  4. Jones, R.B., Gordus, A., Krall, J.A. & MacBeath, G. A quantitative protein interaction network for the ErbB receptors using protein microarrays. Nature 439, 168–174 (2006).

    Article  CAS  Google Scholar 

  5. Weiss, W.A., Taylor, S.S. & Shokat, K.M. Recognizing and exploiting differences between RNAi and small-molecule inhibitors. Nat. Chem. Biol. 3, 739–744 (2007).

    Article  CAS  Google Scholar 

  6. Visintin, M., Melchionna, T., Cannistraci, I. & Cattaneo, A. In vivo selection of intrabodies specifically targeting protein-protein interactions: a general platform for an “undruggable” class of disease targets. J. Biotechnol. 135, 1–15 (2008).

    Article  CAS  Google Scholar 

  7. Campbell, S.J. & Jackson, R.M. Diversity in the SH2 domain family phosphotyrosyl peptide binding site. Protein Eng. 16, 217–227 (2003).

    Article  CAS  Google Scholar 

  8. Bradshaw, J.M., Mitaxov, V. & Waksman, G. Investigation of phosphotyrosine recognition by the SH2 domain of the Src kinase. J. Mol. Biol. 293, 971–985 (1999).

    Article  CAS  Google Scholar 

  9. Ladbury, J.E. & Arold, S. Searching for specificity in SH domains. Chem. Biol. 7, R3–R8 (2000).

    Article  CAS  Google Scholar 

  10. Taylor, J.D. et al. Structure, dynamics, and binding thermodynamics of the v-Src SH2 domain: implications for drug design. Proteins 73, 929–940 (2008).

    Article  CAS  Google Scholar 

  11. Gan, W. & Roux, B. Binding specificity of SH2 domains: insight from free energy simulations. Proteins 74, 996–1007 (2009).

    Article  CAS  Google Scholar 

  12. Shakespeare, W.C. SH2 domain inhibition: a problem solved? Curr. Opin. Chem. Biol. 5, 409–415 (2001).

    Article  CAS  Google Scholar 

  13. Machida, K. & Mayer, B.J. The SH2 domain: versatile signaling module and pharmaceutical target. Biochim. Biophys. Acta 1747, 1–25 (2005).

    Article  CAS  Google Scholar 

  14. Mandine, E. et al. High-affinity Src-SH2 ligands which do not activate Tyr(527)-phosphorylated Src in an experimental in vivo system. Biochem. Biophys. Res. Commun. 298, 185–192 (2002).

    Article  CAS  Google Scholar 

  15. Garcia-Echeverria, C. Antagonists of the Src homology 2 (SH2) domains of Grb2, Src, Lck and ZAP-70. Curr. Med. Chem. 8, 1589–1604 (2001).

    Article  CAS  Google Scholar 

  16. Reichert, J.M., Rosensweig, C.J., Faden, L.B. & Dewitz, M.C. Monoclonal antibody successes in the clinic. Nat. Biotechnol. 23, 1073–1078 (2005).

    Article  CAS  Google Scholar 

  17. Koide, A., Bailey, C.W., Huang, X. & Koide, S. The fibronectin type III domain as a scaffold for novel binding proteins. J. Mol. Biol. 284, 1141–1151 (1998).

    Article  CAS  Google Scholar 

  18. Koide, A., Abbatiello, S., Rothgery, L. & Koide, S. Probing protein conformational changes in living cells by using designer binding proteins: application to the estrogen receptor. Proc. Natl. Acad. Sci. USA 99, 1253–1258 (2002).

    Article  CAS  Google Scholar 

  19. Gilbreth, R.N., Esaki, K., Koide, A., Sidhu, S.S. & Koide, S. A dominant conformational role for amino acid diversity in minimalist protein-protein interfaces. J. Mol. Biol. 381, 407–418 (2008).

    Article  CAS  Google Scholar 

  20. Daley, G.Q., Van Etten, R.A. & Baltimore, D. Induction of chronic myelogenous leukemia in mice by the P210bcr/abl gene of the Philadelphia chromosome. Science 247, 824–830 (1990).

    Article  CAS  Google Scholar 

  21. Nagar, B. et al. Organization of the SH3–SH2 unit in active and inactive forms of the c-Abl tyrosine kinase. Mol. Cell 21, 787–798 (2006).

    Article  CAS  Google Scholar 

  22. Hantschel, O. & Superti-Furga, G. Regulation of the c-Abl and Bcr-Abl tyrosine kinases. Nat. Rev. Mol. Cell Biol. 5, 33–44 (2004).

    Article  CAS  Google Scholar 

  23. Filippakopoulos, P. et al. Structural coupling of SH2-kinase domains links Fes and Abl substrate recognition and kinase activation. Cell 134, 793–803 (2008).

    Article  CAS  Google Scholar 

  24. Hackel, B.J., Kapila, A. & Wittrup, K.D. Picomolar affinity fibronectin domains engineered utilizing loop length diversity, recursive mutagenesis, and loop shuffling. J. Mol. Biol. 381, 1238–1252 (2008).

    Article  CAS  Google Scholar 

  25. Tokonzaba, E., Capelluto, D.G., Kutateladze, T.G. & Overduin, M. Phosphoinositide, phosphopeptide and pyridone interactions of the Abl SH2 domain. Chem. Biol. Drug Des. 67, 230–237 (2006).

    Article  CAS  Google Scholar 

  26. Porter, C.J. et al. Grb7 SH2 domain structure and interactions with a cyclic peptide inhibitor of cancer cell migration and proliferation. BMC Struct. Biol. 7, 58 (2007).

    Article  Google Scholar 

  27. Burckstummer, T. et al. An efficient tandem affinity purification procedure for interaction proteomics in mammalian cells. Nat. Methods 3, 1013–1019 (2006).

    Article  Google Scholar 

  28. Brehme, M. et al. Charting the molecular network of the drug target Bcr-Abl. Proc. Natl. Acad. Sci. USA 106, 7414–7419 (2009).

    Article  CAS  Google Scholar 

  29. Basu, D., El-Assal Sel, D., Le, J., Mallery, E.L. & Szymanski, D.B. Interchangeable functions of Arabidopsis PIROGI and the human WAVE complex subunit SRA1 during leaf epidermal development. Development 131, 4345–4355 (2004).

    Article  CAS  Google Scholar 

  30. Ewing, R.M. et al. Large-scale mapping of human protein-protein interactions by mass spectrometry. Mol. Syst. Biol. 3, 89 (2007).

    Article  Google Scholar 

  31. Wu, C. et al. Systematic identification of SH3 domain-mediated human protein-protein interactions by peptide array target screening. Proteomics 7, 1775–1785 (2007).

    Article  CAS  Google Scholar 

  32. MacPartlin, M., Smith, A.M., Druker, B.J., Honigberg, L.A. & Deininger, M.W. Bruton's tyrosine kinase is not essential for Bcr-Abl–mediated transformation of lymphoid or myeloid cells. Leukemia 22, 1354–1360 (2008).

    Article  CAS  Google Scholar 

  33. Pendergast, A.M. et al. BCR-ABL-induced oncogenesis is mediated by direct interaction with the SH2 domain of the GRB-2 adaptor protein. Cell 75, 175–185 (1993).

    Article  CAS  Google Scholar 

  34. Hantschel, O. et al. The chemokine interleukin-8 and the surface activation protein CD69 are markers for Bcr-Abl activity in chronic myeloid leukemia. Mol. Oncol. 2, 272–281 (2008).

    Article  Google Scholar 

  35. El Hader, C. et al. HCaRG increases renal cell migration by a TGF-α autocrine loop mechanism. Am. J. Physiol. Renal Physiol. 289, F1273–F1280 (2005).

    Article  CAS  Google Scholar 

  36. Nagar, B. et al. Structural basis for the autoinhibition of c-Abl tyrosine kinase. Cell 112, 859–871 (2003).

    Article  CAS  Google Scholar 

  37. Overduin, M., Rios, C.B., Mayer, B.J., Baltimore, D. & Cowburn, D. Three-dimensional solution structure of the src homology 2 domain of c-abl. Cell 70, 697–704 (1992).

    Article  CAS  Google Scholar 

  38. Waksman, G., Shoelson, S.E., Pant, N., Cowburn, D. & Kuriyan, J. Binding of a high affinity phosphotyrosyl peptide to the Src SH2 domain: crystal structures of the complexed and peptide-free forms. Cell 72, 779–790 (1993).

    Article  CAS  Google Scholar 

  39. Eck, M.J., Shoelson, S.E. & Harrison, S.C. Recognition of a high-affinity phosphotyrosyl peptide by the Src homology-2 domain of p56lck. Nature 362, 87–91 (1993).

    Article  CAS  Google Scholar 

  40. Lawrence, M.C. & Colman, P.M. Shape complementarity at protein/protein interfaces. J. Mol. Biol. 234, 946–950 (1993).

    Article  CAS  Google Scholar 

  41. Hantschel, O. et al. A myristoyl/phosphotyrosine switch regulates c-Abl. Cell 112, 845–857 (2003).

    Article  CAS  Google Scholar 

  42. Patwardhan, P. & Miller, W.T. Processive phosphorylation: mechanism and biological importance. Cell. Signal. 19, 2218–2226 (2007).

    Article  CAS  Google Scholar 

  43. Schaller, M.D. & Schaefer, E.M. Multiple stimuli induce tyrosine phosphorylation of the Crk-binding sites of paxillin. Biochem. J. 360, 57–66 (2001).

    Article  CAS  Google Scholar 

  44. Mayer, B.J., Hirai, H. & Sakai, R. Evidence that SH2 domains promote processive phosphorylation by protein-tyrosine kinases. Curr. Biol. 5, 296–305 (1995).

    Article  CAS  Google Scholar 

  45. Ye, D., Wolff, N., Li, L., Zhang, S. & Ilaria, R.L. Jr. STAT5 signaling is required for the efficient induction and maintenance of CML in mice. Blood 107, 4917–4925 (2006).

    Article  CAS  Google Scholar 

  46. Nieborowska-Skorska, M. et al. Signal transducer and activator of transcription (STAT)5 activation by BCR/ABL is dependent on intact Src homology (SH)3 and SH2 domains of BCR/ABL and is required for leukemogenesis. J. Exp. Med. 189, 1229–1242 (1999).

    Article  CAS  Google Scholar 

  47. Schoepfer, J. et al. Highly potent inhibitors of the Grb2–SH2 domain. Bioorg. Med. Chem. Lett. 9, 221–226 (1999).

    Article  CAS  Google Scholar 

  48. Poy, F. et al. Crystal structures of the XLP protein SAP reveal a class of SH2 domains with extended, phosphotyrosine-independent sequence recognition. Mol. Cell 4, 555–561 (1999).

    Article  CAS  Google Scholar 

  49. Bae, J.H. et al. The selectivity of receptor tyrosine kinase signaling is controlled by a secondary SH2 domain binding site. Cell 138, 514–524 (2009).

    Article  CAS  Google Scholar 

  50. Fellouse, F.A. et al. High-throughput generation of synthetic antibodies from highly functional minimalist phage-displayed libraries. J. Mol. Biol. 373, 924–940 (2007).

    Article  CAS  Google Scholar 

  51. Koide, A., Gilbreth, R.N., Esaki, K., Tereshko, V. & Koide, S. High-affinity single-domain binding proteins with a binary-code interface. Proc. Natl. Acad. Sci. USA 104, 6632–6637 (2007).

    Article  CAS  Google Scholar 

  52. Fellouse, F.A. et al. Molecular recognition by a binary code. J. Mol. Biol. 348, 1153–1162 (2005).

    Article  CAS  Google Scholar 

  53. Fellouse, F.A., Barthelemy, P.A., Kelley, R.F. & Sidhu, S.S. Tyrosine plays a dominant functional role in the paratope of a synthetic antibody derived from a four amino acid code. J. Mol. Biol. 357, 100–114 (2006).

    Article  CAS  Google Scholar 

  54. Hoppe-Seyler, F., Crnkovic-Mertens, I., Tomai, E. & Butz, K. Peptide aptamers: specific inhibitors of protein function. Curr. Mol. Med. 4, 529–538 (2004).

    Article  CAS  Google Scholar 

  55. Mayer, B.J., Jackson, P.K., Van Etten, R.A. & Baltimore, D. Point mutations in the abl SH2 domain coordinately impair phosphotyrosine binding in vitro and transforming activity in vivo. Mol. Cell. Biol. 12, 609–618 (1992).

    Article  CAS  Google Scholar 

  56. Skorski, T. et al. Transformation of hematopoietic cells by BCR/ABL requires activation of a PI-3k/Akt-dependent pathway. EMBO J. 16, 6151–6161 (1997).

    Article  CAS  Google Scholar 

  57. Koide, A. & Koide, S. Monobodies: antibody mimics based on the scaffold of the fibronectin type III domain. Methods Mol. Biol. 352, 95–109 (2007).

    CAS  PubMed  Google Scholar 

  58. Machida, K. et al. High-throughput phosphotyrosine profiling using SH2 domains. Mol. Cell 26, 899–915 (2007).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank R. Gilbreth and E. Duguid for assistance with X-ray structure determination, E. Duguid and C. He for assistance with DNA synthesis, M. Ciaccio, R. Gilbreth, P. Nash, B. Liu and A.A. Kossiakoff for discussion, the staff of the Life Sciences Collaborative Access Team (LS-CAT) beamline at the Advanced Photon Source and the University of Chicago DNA Sequencing Core facility for technical support, A.A. Kossiakoff for access to the BIAcore instrument, A. MĂĽller, N. Venturini and M. Planyavsky for expert assistance with the MS analysis and the Structural Genomics Consortium for making the SH2 vectors available through Open Biosystems. This work was supported by US National Institutes of Health grants R01-GM72688, U54-GM74946 and R21-CA132700 to S.K., by the University of Chicago Cancer Research Center and by the Austrian Academy of Sciences. J.W. was supported in part by US National Institutes of Health grant 5T32GM07281-33 and European Molecular Biology Organization fellowship ASTF 293.00-2009. Use of the Advanced Photon Source was supported by the US Department of Energy, Office of Science, Office of Basic Energy Sciences under Contract No. DE-AC02-06CH11357. Use of the LS-CAT Sector 21 was supported by the Michigan Economic Development Corporation and the Michigan Technology Tri-Corridor for the support of this research program (grant 085P1000817).

Author information

Authors and Affiliations

  1. Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, Illinois, USA

    John Wojcik, Akiko Koide & Shohei Koide

  2. Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria

    Oliver Hantschel, Florian Grebien, Ines Kaupe, Keiryn L Bennett & Giulio Superti-Furga

  3. Ben May Department for Cancer Research, University of Chicago, Chicago, Illinois, USA

    John Barkinge & Richard B Jones

  4. Institute for Genomics and Systems Biology, University of Chicago, Chicago, Illinois, USA

    John Barkinge & Richard B Jones

Authors
  1. John Wojcik
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Oliver Hantschel
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Florian Grebien
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Ines Kaupe
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Keiryn L Bennett
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. John Barkinge
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Richard B Jones
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Akiko Koide
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Giulio Superti-Furga
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Shohei Koide
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

J.W., A.K. and S.K. designed phage-display library, selection and biophysical characterization; A.K. and J.W. optimized phage-display methods; J.W. performed selections, biophysical characterization, crystallization and structure determination; J.B. and R.B.J. designed and made SH2 microarrays; J.W. and J.B. conducted microarray experiments; J.W., F.G., O.H. and G.S.F. designed cellular experiments and kinase assays; J.W., O.H., F.G. and I.K. conducted cellular studies and kinase assays; K.L.B. conducted MS and data analysis. J.W., O.H., F.G., R.B.J., G.S.F. and S.K. wrote the manuscript.

Corresponding author

Correspondence to Shohei Koide.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1 and 2, Supplementary Tables 1–3, Supplementary Data and Supplementary Methods (PDF 626 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Wojcik, J., Hantschel, O., Grebien, F. et al. A potent and highly specific FN3 monobody inhibitor of the Abl SH2 domain. Nat Struct Mol Biol 17, 519–527 (2010). https://doi.org/10.1038/nsmb.1793

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nsmb.1793

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing