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Structural determinants of integrin β-subunit specificity for latent TGF-β

Abstract

Eight integrin α-β heterodimers recognize ligands with an Arg-Gly-Asp (RGD) motif. However, the structural mechanism by which integrins differentiate among extracellular proteins with RGD motifs is not understood. Here, crystal structures, mutations and peptide-affinity measurements show that αVβ6 binds with high affinity to a RGDLXXL/I motif within the prodomains of TGF-β1 and TGF-β3. The LXXL/I motif forms an amphipathic α-helix that binds in a hydrophobic pocket in the β6 subunit. Elucidation of the basis for ligand binding specificity by the integrin β subunit reveals contributions by three different βI-domain loops, which we designate specificity-determining loops (SDLs) 1, 2 and 3. Variation in a pair of single key residues in SDL1 and SDL3 correlates with the variation of the entire β subunit in integrin evolution, thus suggesting a paradigmatic role in overall β-subunit function.

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Figure 1: Activation and binding of pro-TGF-β1 by wild-type and mutant αV integrins.
Figure 2: Crystal structures and comparisons of the αVβ6 headpiece.
Figure 3: Ligands of αVβ6.

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References

  1. Hynes, R.O. Integrins: bi-directional, allosteric, signaling machines. Cell 110, 673–687 (2002).

    Article  CAS  Google Scholar 

  2. Munger, J.S. et al. The integrin αvβ6 binds and activates latent TGFβ1: a mechanism for regulating pulmonary inflammation and fibrosis. Cell 96, 319–328 (1999).

    Article  CAS  Google Scholar 

  3. Mu, D. et al. The integrin αvβ8 mediates epithelial homeostasis through MT1-MMP-dependent activation of TGF-β1. J. Cell Biol. 157, 493–507 (2002).

    Article  CAS  Google Scholar 

  4. Shull, M.M. et al. Targeted disruption of the mouse transforming growth factor-β1 gene results in multifocal inflammatory disease. Nature 359, 693–699 (1992).

    Article  CAS  Google Scholar 

  5. Yang, Z. et al. Absence of integrin-mediated TGFβ1 activation in vivo recapitulates the phenotype of TGFβ1-null mice. J. Cell Biol. 176, 787–793 (2007).

    Article  CAS  Google Scholar 

  6. Takagi, J., Kamata, T., Meredith, J., Puzon-McLaughlin, W. & Takada, Y. Changing ligand specificities of αvβ1 and αvβ3 integrins by swapping a short diverse sequence of the β subunit. J. Biol. Chem. 272, 19794–19800 (1997).

    Article  CAS  Google Scholar 

  7. Xiong, J.P. et al. Crystal structure of the extracellular segment of integrin αVβ3 in complex with an Arg-Gly-Asp ligand. Science 296, 151–155 (2002).

    Article  CAS  Google Scholar 

  8. Xiao, T., Takagi, J., Wang, J.-h., Coller, B.S. & Springer, T.A. Structural basis for allostery in integrins and binding of fibrinogen-mimetic therapeutics. Nature 432, 59–67 (2004).

    Article  CAS  Google Scholar 

  9. Nagae, M. et al. Crystal structure of α5β1 integrin ectodomain: atomic details of the fibronectin receptor. J. Cell Biol. 197, 131–140 (2012).

    Article  CAS  Google Scholar 

  10. Sen, M., Yuki, K. & Springer, T.A. An internal ligand-bound, metastable state of a leukocyte integrin, αXβ2. J. Cell Biol. 203, 629–642 (2013).

    Article  CAS  Google Scholar 

  11. Wang, R. et al. GARP regulates the bioavailability and activation of TGF-β. Mol. Biol. Cell 23, 1129–1139 (2012).

    Article  CAS  Google Scholar 

  12. Ludbrook, S.B., Barry, S.T., Delves, C.J. & Horgan, C.M. The integrin αvβ3 is a receptor for the latency-associated peptides of transforming growth factors β1 and β3. Biochem. J. 369, 311–318 (2003).

    Article  CAS  Google Scholar 

  13. Xiong, J.-P. et al. Crystal structure of the extracellular segment of integrin αVβ3. Science 294, 339–345 (2001).

    Article  CAS  Google Scholar 

  14. Zhu, J. et al. Structure of a complete integrin ectodomain in a physiologic resting state and activation and deactivation by applied forces. Mol. Cell 32, 849–861 (2008).

    Article  CAS  Google Scholar 

  15. Dong, X. et al. αVβ3 integrin crystal structures and their functional implications. Biochemistry 51, 8814–8828 (2012).

    Article  CAS  Google Scholar 

  16. Zhu, J., Zhu, J. & Springer, T.A. Complete integrin headpiece opening in eight steps. J. Cell Biol. 201, 1053–1068 (2013).

    Article  CAS  Google Scholar 

  17. Paroutis, P., Touret, N. & Grinstein, S. The pH of the secretory pathway: measurement, determinants, and regulation. Physiology (Bethesda) 19, 207–215 (2004).

    CAS  Google Scholar 

  18. Shi, M. et al. Latent TGF-β structure and activation. Nature 474, 343–349 (2011).

    Article  CAS  Google Scholar 

  19. Acharya, R. et al. The three-dimensional structure of foot-and-mouth disease virus at 2.9 A resolution. Nature 337, 709–716 (1989).

    Article  CAS  Google Scholar 

  20. Takagi, J., Petre, B.M., Walz, T. & Springer, T.A. Global conformational rearrangements in integrin extracellular domains in outside-in and inside-out signaling. Cell 110, 599–611 (2002).

    Article  CAS  Google Scholar 

  21. Springer, T.A., Zhu, J. & Xiao, T. Structural basis for distinctive recognition of fibrinogen γC peptide by the platelet integrin αIIbβ3 . J. Cell Biol. 182, 791–800 (2008).

    Article  CAS  Google Scholar 

  22. Parise, L.V., Helgerson, S.L., Steiner, B., Nannizzi, L. & Phillips, D.R. Synthetic peptides derived from fibrinogen and fibronectin change the conformation of purified platelet glycoprotein IIb-IIIa. J. Biol. Chem. 262, 12597–12602 (1987).

    CAS  PubMed  Google Scholar 

  23. Frelinger, A.L. et al. Occupancy of an adhesive glycoprotein receptor modulates expression of an antigenic site involved in cell adhesion. J. Biol. Chem. 263, 12397–12402 (1988).

    CAS  PubMed  Google Scholar 

  24. Plow, E.F., Pierschbacher, M.D., Ruoslahti, E., Marguerie, G.A. & Ginsberg, M.H. The effect of Arg-Gly-Asp-containing peptides on fibrinogen and von Willebrand factor binding to platelets. Proc. Natl. Acad. Sci. USA 82, 8057–8061 (1985).

    Article  CAS  Google Scholar 

  25. Mas-Moruno, C., Rechenmacher, F. & Kessler, H. Cilengitide: the first anti-angiogenic small molecule drug candidate. Design, synthesis and clinical evaluation. Anticancer Agents Med. Chem. 10, 753–768 (2010).

    Article  CAS  Google Scholar 

  26. Schürpf, T. & Springer, T.A. Regulation of integrin affinity on cell surfaces. EMBO J. 30, 4712–4727 (2011).

    Article  Google Scholar 

  27. Springer, T.A. & Dustin, M.L. Integrin inside-out signaling and the immunological synapse. Curr. Opin. Cell Biol. 24, 107–115 (2012).

    Article  CAS  Google Scholar 

  28. Takagi, J., Debottis, D.P., Erickson, H.P. & Springer, T.A. The role of specificity-determining loop of the integrin β-subunit I-like domain in folding, association with the α subunit, and ligand binding. Biochemistry 41, 4339–4347 (2002).

    Article  CAS  Google Scholar 

  29. Huhtala, M., Heino, J., Casciari, D., de Luise, A. & Johnson, M.S. Integrin evolution: insights from ascidian and teleost fish genomes. Matrix Biol. 24, 83–95 (2005).

    Article  CAS  Google Scholar 

  30. Rossi, A.M. & Taylor, C.W. Analysis of protein-ligand interactions by fluorescence polarization. Nat. Protoc. 6, 365–387 (2011).

    Article  CAS  Google Scholar 

  31. Karplus, P.A. & Diederichs, K. Linking crystallographic model and data quality. Science 336, 1030–1033 (2012).

    Article  CAS  Google Scholar 

  32. Abe, M. et al. An assay for transforming growth factor-β using cells transfected with a plasminogen activator inhibitor-1 promoter-luciferase construct. Anal. Biochem. 216, 276–284 (1994).

    Article  CAS  Google Scholar 

  33. Yu, Y., Schurpf, T. & Springer, T.A. How natalizumab binds and antagonizes α4 integrins. J. Biol. Chem. 288, 32314–32325 (2013).

    Article  CAS  Google Scholar 

  34. Kabsch, W. in International Tables for Crystallography Vol. F (eds Rossmann, M.G. & Arnold, E.) Ch. 25.2.9, 730–734 (Kluwer Academic Publishers, 2001).

  35. McCoy, A.J. et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007).

    Article  CAS  Google Scholar 

  36. Adams, P.D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D Biol. Crystallogr. 66, 213–221 (2010).

    Article  CAS  Google Scholar 

  37. Davis, I.W. et al. MolProbity: all-atom contacts and structure validation for proteins and nucleic acids. Nucleic Acids Res. 35, W375–W383 (2007).

    Article  Google Scholar 

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Acknowledgements

This work was supported by US National Institutes of Health grant NIH P01HL103526 (T.A.S.).

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Authors and Affiliations

Authors

Contributions

X.D. contributed to research design, carried out experiments, analyzed data and wrote the manuscript. N.E.H. helped to analyze the data and prepare the manuscript. C.L. contributed research design. T.A.S. conceived the experimental design, analyzed the data and wrote the manuscript.

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Correspondence to Timothy A Springer.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Omit map of the pro-TGF-β3 peptide.

Simulated annealing composite omit map contoured at 1 σ within 2 Å of the ligand. Carbon color codes are αV, green; β6, cyan; ligand, magenta. Metal ions are silver spheres. The complex shown contains chains A, B, and E.

Supplementary information

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Supplementary Figure 1 and Supplementary Table 1 (PDF 331 kb)

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Dong, X., Hudson, N., Lu, C. et al. Structural determinants of integrin β-subunit specificity for latent TGF-β. Nat Struct Mol Biol 21, 1091–1096 (2014). https://doi.org/10.1038/nsmb.2905

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