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Natural and Artificial Mutations in αIIb Integrin Lead to a Structural Deformation of a Calcium-Binding Site

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Abstract

The platelet integrin αIIbβ3 is widely accepted as a structural and a functional model of the broad integrin protein family. The four calcium-binding sites in the αIIb subunit contribute to biogenesis and stability of the protein. Mansour et al. (J Thromb Haemost 9:192–200, 2011) showed that the natural Asn2Asp mutation causing Glanzmann thrombasthenia, prevented surface expression of αIIbβ3, whereas the artificial Asn2Gln mutation only decreased its level. Molecular dynamics simulations and EDTA chelation assay were used here to explore the mechanism of these structural deformations. We show a considerable expansion of the calcium-binding site 3 in Asn2Asp mutation, whereas the Asn2Gln toggles between normal and expanded conformations. The αIIbβ3 surface expression level correlates to the relative spending time in the expanded conformation. By a comparison to other calcium-binding sites of αIIb and of other α integrins we show that the size of a calcium-binding loop is conserved. EDTA chelation assay shows a sensitivity to calcium removal, which correlates with the reduction in αIIbβ3 surface expression and with the calcium binding site expansion, thus verifying the simulation data. Here we indicate that Asn2 mutation affects a calcium-binding site 3 of αIIb, which structural deformation is proposed to deprive calcium binding and interfere with an integrin intracellular trafficking and its surface expression.

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Abbreviations

LIBS:

Ligand-induced binding sites

GT:

Glanzmann thrombasthenia

References

  1. Springer TA (1997) Folding of the N-terminal, ligand-binding region of integrin alpha-subunits into a beta-propeller domain. Proc Natl Acad Sci USA 94:65–72

    Article  CAS  Google Scholar 

  2. Chen CK-M, Chan N-L, Wang AH-J (2011) The many blades of the β-propeller proteins: conserved but versatile. Trends Biochem Sci 36:553–561

    Article  CAS  Google Scholar 

  3. Murzin AG (1992) Structural principles for the propeller assembly of beta-sheets: the preference for seven-fold symmetry. Proteins 14:191–201

    Article  CAS  Google Scholar 

  4. Xiong JP, Stehle T, Diefenbach B, Zhang R, Dunker R, Scott DL, Joachimiak A, Goodman SL, Arnaout MA (2001) Crystal structure of the extracellular segment of integrin avb3. Science 294:339–345

    Article  CAS  Google Scholar 

  5. Xiao T, Takagi J, Coller BS, Wang JH, Springer T (2004) Structural basis for allostery in integrins and binding to fibrinogen-mimetic therapeutics. Nature 432:59–67

    Article  CAS  Google Scholar 

  6. Zhang K, Chen J (2012) The regulation of integrin function by divalent cations. Cell Adhes Migr 6:20–29

    Article  Google Scholar 

  7. Zhu J, Luo B-H, Xiao T, Zhang C, Nishida N, Springer TA (2008) Structure of a complete integrin ectodomain in a physiologic resting state and activation and deactivation by applied forces. Mol Cell 32:849–861

    Article  CAS  Google Scholar 

  8. Tiwari S, Askari JA, Humphries MJ, Bulleid NJ (2011) Divalent cations regulate the folding and activation status of integrins during their intracellular trafficking. J Cell Sci 124:1672–1680

    Article  CAS  Google Scholar 

  9. Kirchhofer D, Grzesiak J, Pierschbacher MD (1991) Calcium as a potential physiological regulator of integrin-mediated cell adhesion. J Biol Chem 266:4471–4477

    CAS  Google Scholar 

  10. Honda S, Tomiyama Y, Pelletier AJ, Annis D, Honda Y, Orchekowski R, Ruggeri Z, Kunicki TJ (1995) Topography of ligand-induced binding sites, including a novel cation-sensitive epitope (AP5) at the amino terminus, of the human integrin beta 3 subunit. J Biol Chem 270:11947–11954

    Article  CAS  Google Scholar 

  11. Pelletier AJ, Kunicki T, Quaranta V (1996) Activation of the integrin alpha v beta 3 involves a discrete cation-binding site that regulates conformation. J Biol Chem 271:1364–1370

    Article  CAS  Google Scholar 

  12. Gahmberg CG, Fagerholm SC, Nurmi SM, Chavakis T, Marchesan S, Grönholm M (2009) Regulation of integrin activity and signalling. Biochim Biophys Acta 1790:431–444

    Article  CAS  Google Scholar 

  13. Nurden AT, Fiore M, Nurden P, Pillois X (2011) Glanzmann thrombasthenia: a review of ITGA2B and ITGB3 defects with emphasis on variants, phenotypic variability, and mouse models. Blood 118:5996–6005

    Article  CAS  Google Scholar 

  14. Hauschner H, Landau M, Seligsohn U, Rosenberg N (2010) A unique interaction between alphaIIb and beta3 in the head region is essential for outside-in signaling-related functions of alphaIIbbeta3 integrin. Blood 115:4542–4550

    Article  CAS  Google Scholar 

  15. Rosenberg N, Landau M, Luboshitz J, Rechavi G, Seligsohn U (2004) A novel Phe171Cys mutation in integrin alpha causes Glanzmann thrombasthenia by abrogating alphabeta complex formation. J Thromb Haemost 2:1167–1175

    Article  CAS  Google Scholar 

  16. Vijapurkar M, Ghosh K, Shetty S (2009) Novel mutations in GP IIb gene in Glanzmann’s thrombasthenia from India. Platelets 20:35–40

    Article  CAS  Google Scholar 

  17. Pillois X, Fiore M, Heilig R, Pico M, Nurden AT (2013) A novel amino acid substitution of integrin αIIb in Glanzmann thrombasthenia confirms that the N-terminal region of the receptor plays a role in maintaining β-propeller structure. Platelets 24:77–80

    Article  CAS  Google Scholar 

  18. Mansour W, Einav Y, Hauschner H, Koren A, Seligsohn U, Rosenberg N (2011) An αIIb mutation in patients with Glanzmann thrombasthenia located in the N-terminus of blade 1 of the β-propeller (Asn2Asp) disrupts a calcium binding site in blade 6. J Thromb Haemost 9:192–200

    Article  CAS  Google Scholar 

  19. Berendsen HJC, Van Der Spoel D, Van Drunen R (1995) GROMACS: a message-passing parallel molecular dynamics implementation. Comput Phys Commun 91:43–56

    Article  CAS  Google Scholar 

  20. Lindahl E, Hess B, Van Der Spoel D (2001) GROMACS 3.0: a package for molecular simulation and trajectory analysis. J Mol Model 7:306–317

    CAS  Google Scholar 

  21. Van Der Spoel D, Lindahl E, Hess B, Groenhof G, Mark AE, Berendsen HJ (2005) GROMACS: fast, flexible, and free. J Comput Chem 26:1701–1718

    Article  Google Scholar 

  22. Oostenbrink C, Villa A, Mark AE, Van Gunsteren WF (2004) A biomolecular force field based on the free enthalpy of hydration and solvation: the GROMOS force-field parameter sets 53A5 and 53A6. J Comput Chem 25:1656–1676

    Article  CAS  Google Scholar 

  23. Guex N, Peitsch MC (1997) SWISS-MODEL and the Swiss-PDB viewer: an environment for comparative protein modeling. Electrophoresis 18:2714–2723

    Article  CAS  Google Scholar 

  24. Berendsen HJC, Postma JPM, Van Gunsteren WF, Hermans J (1981) Interaction models for water in relation to protein hydration. Intermol Forces 11:331–338

    Article  Google Scholar 

  25. Hess B, Bekker H, Berendsen HJC, Fraaije JGEM (1997) LINCS: a linear constraint solver for molecular simulations. J Comput Chem 18:1463–1472

    Article  CAS  Google Scholar 

  26. Miyamoto S, Kollman PA (1992) SETTLE: an analytical version of the SHAKE and RATTLE algorithm for rigid water models. J Comput Chem 13:952–962

    Article  CAS  Google Scholar 

  27. Berendsen HJC, Postma JPM, Van Gunsteren WF, DiNola A, Haak JR (1984) Molecular dynamics with coupling to an external bath. J Chem Phys 81:3684–3690

    Article  CAS  Google Scholar 

  28. Darden T, York D, Pedersen L (1993) Particle mesh Ewald: an N·log(N) method for Ewald sums in large systems. J Chem Phys 98:10089–10092

    Article  CAS  Google Scholar 

  29. Fitzgerald LA, Phillips DR (1985) Calcium regulation of the platelet membrane glycoprotein IIb-IIIa complex. J Biol Chem 260:211–217

    Google Scholar 

  30. Gachet C, Hanau D, Spehner D, Brisson C, Garaud JC, Schmitt DA, Olflmann P, Cazenave JP (1993) Alpha IIb beta 3 integrin dissociation induced by EDTA results in morphological changes of the platelet surface-connected canalicular system with differential location of the two separate subunits. J Cell Biol 120:1021–1030

    Article  CAS  Google Scholar 

  31. Harding MM (1999) The geometry of metal-ligand interactions relevant to proteins. Acta Crystallogr Sect D Biol Crystallogr 55:1432–1443

    Article  CAS  Google Scholar 

  32. Harding MM (2000) The geometry of metal-ligand interactions relevant to proteins. II. Angles at the metal atom, additional weak metal-donor interactions. Acta Crystallogr Sect D Biol Crystallogr 56:857–867

    Article  CAS  Google Scholar 

  33. Project E, Nachliel E, Gutman M (2008) Parameterization of Ca+2-protein interactions for molecular dynamics simulations. J Comput Chem 29:1163–1169

    Article  Google Scholar 

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Author notes

  1. Wissam Mansour

    Present address: Department of Biology, Technion-Israel Institute of Technology, 32000, Haifa, Israel

Authors and Affiliations

  1. The Amalia Biron Research Institute of Thrombosis and Haemostasis, Chaim Sheba Medical Center, 52621, Tel-Hashomer, Israel

    Wissam Mansour, Hagit Hauschner, Uri Seligsohn & Nurit Rosenberg

  2. Sackler Faculty of Medicine, Tel Aviv University, 69978, Tel Aviv, Israel

    Wissam Mansour, Hagit Hauschner, Uri Seligsohn & Nurit Rosenberg

  3. Mathematical Biology Unit, Faculty of Sciences, Holon Institute of Technology, 58102, Holon, Israel

    Yulia Einav

Authors
  1. Wissam Mansour
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  2. Hagit Hauschner
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  3. Uri Seligsohn
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  4. Nurit Rosenberg
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  5. Yulia Einav
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Corresponding author

Correspondence to Yulia Einav.

Electronic supplementary material

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10930_2014_9579_MOESM1_ESM.tif

Figure S1. Root Mean Square Deviations of the simulated structures. Root Mean Square Deviation values (nm) of the backbone atoms from their initial coordinates are shown as a function of simulation time. Black line – WT, dark gray line – Asn2Asp mutation, light gray line – Asn2Gln mutation. (TIFF 187 kb)

10930_2014_9579_MOESM2_ESM.tif

Figure S2. The level of integrin β3. Cell lysates expressing WT, Asn2Asp or Asn2Gln αIIb were subjected to SDS-PAGE in reduced conditions, following immuno-blotting with monoclonal antibody AP5 against integrin β3. Relative amount of β3 in the WT and the two mutants calculated by densitometry. (TIFF 17 kb)

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Mansour, W., Hauschner, H., Seligsohn, U. et al. Natural and Artificial Mutations in αIIb Integrin Lead to a Structural Deformation of a Calcium-Binding Site. Protein J 33, 474–483 (2014). https://doi.org/10.1007/s10930-014-9579-5

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