Orientation and surface coverage of adsorbed fibronectin cell binding domains and bound integrin α5β1 receptors

Michaela Kreiner, Chandramouli R. Chillakuri, Patricia Pereira, Michael Fairhead, Zhaohui Li, H.J. Mardon, Stephen A. Holt, Christopher F. van der Walle

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Abstract

Integrin α5β1 binds the human 9th-10th type III fibronectin domain pair (FIII9-10) to mediate cell attachment and spreading. FIII9-10 mutants with increased conformational stability (FIII9010) or highly restricted interdomain mobility (FIII9010-CC) support cell spreading to greater and lesser extents, respectively. We have used neutron reflectivity to show that the surface adsorbed layers of the wild-type and mutant proteins are remarkably different. At bulk concentrations of protein equivalent to those used in cell spreading assays, the surface coverage of FIII9-10 was 14% compared to 31% for FIII9010 and 100% for FIII9010-CC. For FIII9010-CC, three distinct transitions in the packing and orientation of the domain pair were observed. No similar transitions were observed for FIII9-10 and only a transition to bilayer was observed for FIII9010. We discuss these observations by analogy to the surface pressure area isotherm of surfactants, with reference to the electrostatic surface potentials and conformational stabilities of the domain pairs. Data for the binding of purified integrin α5β1 receptors to adsorbed FIII9010-CC were fitted with an integrin layer thickness of 130 A ° . This indicates a movement of the integrin α5β1 headpiece away from its position in the compact 'bent' conformation. Thus, neutron reflectivity should prove to be a useful technique for the determination of the averaged integrin conformation upon binding to various ligands.
Original languageEnglish
Pages (from-to)3954-3962
Number of pages8
JournalSoft Matter
Volume5
DOIs
Publication statusPublished - 2009

Keywords

  • orientation
  • adsorbed fibronectin cells
  • binding domains
  • bound integrin α5β1 receptors
  • pharmacology
  • soft matter

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    Kreiner, M., Chillakuri, C. R., Pereira, P., Fairhead, M., Li, Z., Mardon, H. J., Holt, S. A., & van der Walle, C. F. (2009). Orientation and surface coverage of adsorbed fibronectin cell binding domains and bound integrin α5β1 receptors. Soft Matter, 5, 3954-3962. https://doi.org/10.1039/b908706k