Structure of integrin alpha5beta1 in complex with fibronectin

Abstract

The membrane-distal headpiece of integrins has evolved to specifically bind large extracellular protein ligands, but the molecular architecture of the resulting complexes has not been determined. We used molecular electron microscopy to determine the three-dimensional structure of the ligand-binding headpiece of integrin alpha5beta1 complexed with fragments of its physiological ligand fibronectin. The density map for the unliganded alpha5beta1 headpiece shows a 'closed' conformation similar to that seen in the alphaVbeta3 crystal structure. By contrast, binding to fibronectin induces an 'open' conformation with a dramatic, approximately 80 degrees change in the angle of the hybrid domain of the beta subunit relative to its I-like domain. The fibronectin fragment binds to the interface between the beta-propeller and I-like domains in the integrin headpiece through the RGD-containing module 10, but direct contact of the synergy-region-containing module 9 to integrin is not evident. This finding is corroborated by kinetic analysis of real-time binding data, which shows that the synergy site greatly enhances k(on) but has little effect on the stability or k(off) of the complex.

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).

Fig. 2. Projection averages of the α₅β₁ headpiece obtained by negative stain EM. The unclasped α₅β₁ headpiece was incubated (A) without or (B) with 1 mM RGD peptide and imaged in the EM. In the +RGD condition, particles with closed (group I) and open (group II) conformations were observed. The three most typical averages for each group are shown, each containing 200–600 particles. Scale bars, 100 Å.

Fig. 3. Two different integrin headpiece conformations. A representative projection average from (A) unliganded and (D) liganded α₅β₁ headpiece was used to identify the best-correlating projections calculated from a 25 Å density map created from α_Vβ₃ headpiece models (B and E). The model for the open α_Vβ₃ headpiece was prepared as described in the text and Materials and methods section. The models are shown in CPK representation (C and F) using the same color code as in Figure 1A.

Fig. 4. α₅β₁ head in complex with Fn fragment. Projection averages are shown for (A) Fn7–10 alone, (B) α₅β₁ complex with Fn7–10 and (C) α₅β₁ complex with Fn9–10. The projection averages of the α₅β₁/Fn7–10 complexes are subgrouped (groups I, II and III) based on the orientation of the bound ligand, and several representative raw images are shown below (raw). As in Figure 2, the three most populated averages for each group are shown. The assignment of the FnIII modules (black ovals) in the complex is shown in the schematic drawing to the right. The modules missing in the averages due to linker flexibility are depicted by gray ovals. Scale bars, 100 Å.

Fig. 5. Surface-rendered density maps of the α₅β₁ headpiece in (A) the unliganded closed and (B) the ligand-bound open conformation. The unmodified headpiece segments of the α_Vβ₃ crystal structure or the open α_Vβ₃ model were manually fitted into the 3D density map of the unliganded and ligand-bound α₅β₁ headpiece, respectively. Cα worm tracings for α_V, β₃ and Fn10 segments are colored in red, blue and white, respectively. Views are successive 45° rotations about the vertical figure axis. The figure was generated using DINO (Philippsen, 2002).

Fig. 6. Comparison of the kinetics of α₅β₁ binding to recombinant Fn7–10 fragment. Either wild-type, R1379A or RPR/AAA synergy mutant Fn7–10 fragments were immobilized on the sensor chip at the same density (500 RU), and full-length α₅β₁ was injected at 20 µl/min. Traces show increasing concentrations (7.5, 15, 30 and 60 nM) of α₅β₁ analyte.