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Structural basis for allostery in integrins and binding to fibrinogen-mimetic therapeutics

Abstract

Integrins are important adhesion receptors in all Metazoa that transmit conformational change bidirectionally across the membrane. Integrin α and β subunits form a head and two long legs in the ectodomain and span the membrane. Here, we define with crystal structures the atomic basis for allosteric regulation of the conformation and affinity for ligand of the integrin ectodomain, and how fibrinogen-mimetic therapeutics bind to platelet integrin αIIbβ3. Allostery in the β3 I domain alters three metal binding sites, associated loops and α1- and α7-helices. Piston-like displacement of the α7-helix causes a 62° reorientation between the β3 I and hybrid domains. Transmission through the rigidly connected plexin/semaphorin/integrin (PSI) domain in the upper β3 leg causes a 70 Å separation between the knees of the α and β legs. Allostery in the head thus disrupts interaction between the legs in a previously described low-affinity bent integrin conformation, and leg extension positions the high-affinity head far above the cell surface.

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Figure 1: Quaternary rearrangements in the integrin ectodomain.
Figure 2: Structure of the αIIbβ3 headpiece.
Figure 3: The binding sites for ligand-mimetic antagonists and fibrinogen at the α/β subunit interface.
Figure 4: Allostery in the β I domain and comparison with α I domain.
Figure 5: The hybrid and PSI domains and their interfaces.

References

  1. Hughes, P. E. & Pfaff, M. Integrin affinity modulation. Trends Cell Biol. 8, 359–364 (1998)

    CAS  Article  Google Scholar 

  2. Takagi, J. & Springer, T. A. Integrin activation and structural rearrangement. Immunol. Rev. 186, 141–163 (2002)

    CAS  Article  Google Scholar 

  3. 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)

    CAS  Article  Google Scholar 

  4. Springer, T. A. & Wang, J.-h. in Cell Surface Receptors (ed. Garcia, K. C.) (Elsevier, San Diego, 2004)

    Google Scholar 

  5. Coller, B. S. Platelet GPIIb/IIIa antagonists: the first anti-integrin receptor therapeutics. J. Clin. Invest. 99, 1467–1471 (1997)

    CAS  Article  Google Scholar 

  6. Takagi, J., Strokovich, K., Springer, T. A. & Walz, T. Structure of integrin α5β1 in complex with fibronectin. EMBO J. 22, 4607–4615 (2003)

    CAS  Article  Google Scholar 

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

    ADS  CAS  Article  Google Scholar 

  8. 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)

    ADS  CAS  Article  Google Scholar 

  9. Luo, B.-H., Springer, T. A. & Takagi, J. Stabilizing the open conformation of the integrin headpiece with a glycan wedge increases affinity for ligand. Proc. Natl Acad. Sci. USA 100, 2403–2408 (2003)

    ADS  CAS  Article  Google Scholar 

  10. Luo, B.-H., Strokovich, K., Walz, T., Springer, T. A. & Takagi, J. Allosteric β1 integrin antibodies that stabilize the low affinity state by preventing the swing-out of the hybrid domain. J. Biol. Chem. 279, 27466–27471 (2004)

    CAS  Article  Google Scholar 

  11. Luo, B.-H., Springer, T. A. & Takagi, J. High affinity ligand binding by integrins does not involve head separation. J. Biol. Chem. 278, 17185–17189 (2003)

    CAS  Article  Google Scholar 

  12. Mould, A. P. et al. Structure of an integrin-ligand complex deduced from solution X-ray scattering and site-directed mutagenesis. J. Biol. Chem. 278, 39993–39999 (2003)

    CAS  Article  Google Scholar 

  13. Mould, A. P. et al. Conformational changes in the integrin βA domain provide a mechanism for signal transduction via hybrid domain movement. J. Biol. Chem. 278, 17028–17035 (2003)

    CAS  Article  Google Scholar 

  14. Du, X. et al. Ligands “activate” integrin αIIbβ3 (platelet GPIIb-IIIa). Cell 65, 409–416 (1991)

    CAS  Article  Google Scholar 

  15. Adair, B. D. & Yeager, M. Three-dimensional model of the human platelet integrin αIIbβ3 based on electron cryomicroscopy and X-ray crystallography. Proc. Natl Acad. Sci. USA 99, 14059–14064 (2002)

    ADS  CAS  Article  Google Scholar 

  16. Luo, B.-H., Springer, T. A. & Takagi, J. A specific interface between integrin transmembrane helices and affinity for ligand. PLoS Biol. 2, 776–786 (2004)

    CAS  Google Scholar 

  17. Vinogradova, O. et al. A structural mechanism of integrin αIIbβ3 “inside-out” activation as regulated by its cytoplasmic face. Cell 110, 587–597 (2002)

    CAS  Article  Google Scholar 

  18. Kim, M., Carman, C. V. & Springer, T. A. Bidirectional transmembrane signaling by cytoplasmic domain separation in integrins. Science 301, 1720–1725 (2003)

    ADS  CAS  Article  Google Scholar 

  19. Coller, B. S., Peerschke, E. I., Scudder, L. E. & Sullivan, C. A. A murine monoclonal antibody that completely blocks the binding of fibrinogen to platelets produces a thrombasthenic-like state in normal platelets and binds to glycoproteins IIb and/or IIIa. J. Clin. Invest. 72, 325–338 (1983)

    CAS  Article  Google Scholar 

  20. Kamata, T., Tieu, K. K., Springer, T. A. & Takada, Y. Amino acid residues in the αIIb subunit that are critical for ligand binding to integrin αIIbβ3 are clustered in the β-propeller model. J. Biol. Chem. 276, 44275–44283 (2001)

    CAS  Article  Google Scholar 

  21. Artoni, A. et al. The specificity determining loop and α helix 1 on human integrin β3 determine the binding of murine monoclonal antigbody 7E3 to αIIbβ3: implications for the mechanism of integrin activation. Proc. Natl Acad. Sci. USA (in the press) (2004)

  22. Zavortink, M., Bunch, T. A. & Brower, D. L. Functional properties of alternatively spliced forms of the Drosphila PS2 integrin α subunit. Cell Adhes. Commun. 1, 251–264 (1993)

    CAS  Article  Google Scholar 

  23. von der Mark, H. et al. Alternative splice variants of α7β1 integrin selectivity recognize different laminin isoforms. J. Biol. Chem. 277, 6012–6016 (2002)

    CAS  Article  Google Scholar 

  24. Springer, T. A. Predicted and experimental structures of integrins and β-propellers. Curr. Opin. Struct. Biol. 12, 802–813 (2002)

    CAS  Article  Google Scholar 

  25. Lee, J.-O., Rieu, P., Arnaout, M. A. & Liddington, R. Crystal structure of the A domain from the α subunit of integrin CR3 (CD11b/CD18). Cell 80, 631–638 (1995)

    CAS  Article  Google Scholar 

  26. Lee, J.-O., Bankston, L. A., Arnaout, M. A. & Liddington, R. C. Two conformations of the integrin A-domain (I-domain): a pathway for activation? Structure 3, 1333–1340 (1995)

    CAS  Article  Google Scholar 

  27. Shimaoka, M. et al. Structures of the αL I domain and its complex with ICAM-1 reveal a shape-shifting pathway for integrin regulation. Cell 112, 99–111 (2003)

    CAS  Article  Google Scholar 

  28. Scarborough, R. M. & Gretler, D. D. Platelet glycoprotein IIb-IIIa antagonists as prototypical integrin blockers: novel parenteral and potential oral antithrombotic agents. J. Med. Chem. 43, 3453–3473 (2000)

    CAS  Article  Google Scholar 

  29. Gottschalk, K. E. & Kessler, H. The structures of integrins and integrin-ligand complexes: implications for drug design and signal transduction. Angew. Chem. Int. Edn Engl. 41, 3767–3774 (2002)

    CAS  Article  Google Scholar 

  30. Egbertson, M. S. et al. Non-peptide GPIIb/IIIa inhibitors. 20. Centrally constrained thienothiophene alpha-sulfonamides are potent, long acting in vivo inhibitors of platelet aggregation. J. Med. Chem. 42, 2409–2421 (1999)

    CAS  Article  Google Scholar 

  31. Scarborough, R. M. et al. Design of potent and specific integrin antagonists. J. Biol. Chem. 268, 1066–1073 (1993)

    CAS  PubMed  Google Scholar 

  32. Mould, A. P. et al. Integrin activation involves a conformational change in the α1 helix of the β subunit A-domain. J. Biol. Chem. 277, 19800–19805 (2002)

    CAS  Article  Google Scholar 

  33. Chen, J. F., Salas, A. & Springer, T. A. Bistable regulation of integrin adhesiveness by a bipolar metal ion cluster. Nature Struct. Biol. 10, 995–1001 (2003)

    CAS  Article  Google Scholar 

  34. Perutz, M. F. Mechanisms of cooperativity and allosteric regulation in proteins. Q. Rev. Biophys. 22, 139–237 (1989)

    CAS  Article  Google Scholar 

  35. Love, C. A. et al. The ligand-binding face of the semaphorins revealed by the high-resolution crystal structure of SEMA4D. Nature Struct. Biol. 10, 843–848 (2003)

    CAS  Article  Google Scholar 

  36. Bork, P., Doerks, T., Springer, T. A. & Snel, B. Domains in plexins: Links to integrins and transcription factors. Trends Biochem. Sci. 24, 261–263 (1999)

    CAS  Article  Google Scholar 

  37. Calvete, J. J., Henschen, A. & González-Rodríguez, J. Assignment of disulphide bonds in human platelet GPIIIa. A disulphide pattern for the β-subunits of the integrin family. Biochem. J. 274, 63–71 (1991)

    CAS  Article  Google Scholar 

  38. Beglova, N., Blacklow, S. C., Takagi, J. & Springer, T. A. Cysteine-rich module structure reveals a fulcrum for integrin rearrangement upon activation. Nature Struct. Biol. 9, 282–287 (2002)

    CAS  Article  Google Scholar 

  39. Newman, P. J., Derbes, R. S. & Aster, R. H. The human platelet alloantigens, PlA1 and PlA2, are associated with a leucine33/proline33 amino acid polymorphism in membrane glycoprotein IIIa, and are distinguishable by DNA typing. J. Clin. Invest. 83, 1778–1781 (1989)

    CAS  Article  Google Scholar 

  40. Watkins, N. A. et al. HPA-1a phenotype-genotype discrepancy reveals a naturally occurring Arg93Gln substitution in the platelet β3 integrin that disrupts the HPA-1a epitope. Blood 99, 1833–1839 (2002)

    CAS  Article  Google Scholar 

  41. Kunicki, T. J. et al. The P1A alloantigen system is a sensitive indicator of the structural integrity of the amino-terminal domain of the human integrin β3 subunit. Blood Cells Mol. Dis. 21, 131–141 (1995)

    CAS  Article  Google Scholar 

  42. Chen, J. F. et al. The relative influence of metal ion binding sites in the I-like domain and the interface with the hybrid domain on rolling and firm adhesion by integrin α4β7. J. Biol. Chem. (in the press)

  43. Luo, B.-H., Takagi, J. & Springer, T. A. Locking the β3 integrin I-like domain into high and low affinity conformations with disulfides. J. Biol. Chem. 279, 10215–10221 (2004)

    CAS  Article  Google Scholar 

  44. Yang, W., Shimaoka, M., Chen, J. F. & Springer, T. A. Activation of integrin β subunit I-like domains by one-turn C-terminal α-helix deletions. Proc. Natl Acad. Sci. USA 101, 2333–2338 (2004)

    ADS  CAS  Article  Google Scholar 

  45. Takagi, J., Erickson, H. P. & Springer, T. A. C-terminal opening mimics “inside-out” activation of integrin α5β1. Nature Struct. Biol. 8, 412–416 (2001)

    CAS  Article  Google Scholar 

  46. Esnouf, R. M. An extensively modified version of MolScript that includes greatly enhanced coloring capabilities. J. Mol. Graph. Model. 15, 132–138 (1997)

    CAS  Article  Google Scholar 

  47. Merritt, E. A. & Murphy, M. E. P. Raster 3D version 2.0: a program for photorealistic graphics. Acta Crystallogr. D 50, 869–873 (1994)

    CAS  Article  Google Scholar 

  48. Carson, M. Ribbons. Methods Enzymol. 277, 493–505 (1997)

    CAS  Article  Google Scholar 

  49. Puzon-McLaughlin, W., Kamata, T. & Takada, Y. Multiple discontinuous ligand-mimetic antibody binding sites define a ligand binding pocket in integrin αIIbβ3. J. Biol. Chem. 275, 7795–7802 (2000)

    CAS  Article  Google Scholar 

  50. Tozer, E. C., Liddington, R. C., Sutcliffe, M. J., Smeeton, A. H. & Loftus, J. C. Ligand binding to integrin αIIbβ3 is dependent on a MIDAS-like domain in the β3 subunit. J. Biol. Chem. 271, 21978–21984 (1996)

    CAS  Article  Google Scholar 

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Acknowledgements

We thank colleagues in the Springer laboratory for supporting data and stimulating discussions, B. Kessler for tandem mass spectrometry, E. Yvonne Jones at Oxford for sema4D coordinates, members of the J.H.W. and M. Eck laboratories and the staff at APS and CHESS for assistance with crystallography, M. Gerstein and N. Echols (Yale University) for the morphing script used in producing movies, and Y. Cheng for help with comparing crystal structures and the electron microscopy map. Supported by NIH grants to T.A.S., J.H.W. and B.S.C.

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Correspondence to Timothy A. Springer.

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Competing interests

T.A.S. owns shares in Millennium Pharmaceuticals, which markets eptifibatide.

B.S.C. is an inventor of abciximab, an αIIbβ3 antagonist, and in accord with Federal law and the policies of the Research Foundation of the State University

of New York, shares in royalties paid to the Foundation. The rest of the authors declare that they have no competing financial interests.

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Xiao, T., Takagi, J., Coller, B. et al. Structural basis for allostery in integrins and binding to fibrinogen-mimetic therapeutics. Nature 432, 59–67 (2004). https://doi.org/10.1038/nature02976

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