Fig. 1. The truncated α₅β₁ headpiece retains its functional integrity. (A) The domain organization within the primary structure of integrin α₅β₁ is shown on the left, and the design of the recombinant soluble α₅β₁ headpiece is shown on the right. Disulfide-bonded α-helical coiled-coil domains were attached to the C-termini of the truncated subunits to act as a clasp (Takagi et al., 2001). Domains included in the headpiece fragment are color coded as follows: α₅ β-propeller repeat domain in pink, α₅ thigh domain in yellow, β₁ PSI domain in gray, β₁ I-like domain in cyan and β₁ hybrid domain in red. Domains not resolved in the crystal structure are depicted by dotted lines, and the position of the long-range disulfide bond present in the β subunit is shown below. (B) Binding analysis of Fn9–10 to the α₅β₁ head fragment by surface plasmon resonance. Clasped (–TEV, dotted lines) or unclasped (+TEV, solid lines) α₅β₁ headpieces were preincubated with (red lines) or without (black lines) a 3-fold molar excess of TS2/16 Fab fragment for more than 10 min and infused at a concentration of 50 nM onto the sensor surface coated with 650 RU of Fn9–10. Arrows indicate start- and end-points of the injections. (C) Gel filtration chromatography of the α₅β₁ headpiece with bound ligands. The TEV-cleaved α₅β₁ headpiece (∼70 pmol) was incubated with 150 pmol of Fn9–10 (blue), Fn7–10 (red) or without ligands (black) in the presence of 1 mM Mn²⁺ for 1 h and separated on a Superdex 200 column. Chromatograms for 150 pmol Fn7–10 (red dotted) or Fn9–10 (blue dotted) alone are also shown. The elution positions of standard proteins are indicated by arrows (667 kDa, thyroglobulin; 443 kDa, apoferritin; 200 kDa, β-amylase; 67 kDa, serum albumin; 29 kDa, carbonic anhydrase; 12.4 kDa, cytochrome c).