Abstract
The adaptor protein talin serves both to activate the integrin family of cell adhesion molecules and to couple integrins to the actin cytoskeleton. Integrin activation has been shown to involve binding of the talin FERM domain to membrane proximal sequences in the cytoplasmic domain of the integrin β-subunit. However, a second integrin-binding site (IBS2) has been identified near the C-terminal end of the talin rod. Here we report the crystal structure of IBS2 (residues 1974-2293), which comprises two five-helix bundles, “IBS2-A” (1974-2139) and “IBS2-B” (2140-2293), connected by a continuous helix with a distinct kink at its center that is stabilized by side-chain H-bonding. Solution studies using small angle x-ray scattering and NMR point to a fairly flexible quaternary organization. Using pull-down and enzyme-linked immunosorbent assays, we demonstrate that integrin binding requires both IBS2 domains, as does binding to acidic phospholipids and robust targeting to focal adhesions. We have defined the membrane proximal region of the integrin cytoplasmic domain as the major binding region, although more membrane distal regions are also required for strong binding. Alanine-scanning mutagenesis points to an important electrostatic component to binding. Thermal unfolding experiments show that integrin binding induces conformational changes in the IBS2 module, which we speculate are linked to vinculin and membrane binding.
Highlights
1974 –2482 and 1974 –2541, all bound strongly to integrin 3, Vinculin—Talin fragments from the IBS2 region have been whereas the C-terminal domain alone shown to localize to focal adhesions (FAs) [25, 43], but because bound weakly (Fig. 4, A and B)
Talin head (resi- phoinositides in the plasma membrane [5], we explored the dues 1– 405) bound in a dose-dependent manner to 3-integrin binding of IBS2 to a range of phospholipids spotted on a tail (Fig. 4C) with an EC50 of 0.4 Ϯ 0.2 M, consistent with nitrocellulose membrane
We found that IBS2 bound published surface plasmon resonance studies [23]
Summary
Protein Expression and Purification—The cDNAs encoding murine talin residues 1974 –2293, 1974 –2140, and 2137–2293 were synthesized by PCR using a mouse talin cDNA as template and cloned into expression vector pET-151/D-TOPO (Invitrogen). NMR Spectroscopy—NMR spectra were collected using a 0.2 mM protein solution in 20 mM sodium phosphate buffer (pH 6.5), 50 mM NaCl, and 2 mM dithiothreitol at 298 K on a Bruker AVANCE DRX600 spectrometer equipped with a cryoprobe. Wells were incubated with purified recombinant His-Avi-tagged integrin tails (2 g/ml) diluted in PBS containing 1% bovine serum albumin and 0.2% Tween 20 (sample buffer). Phospholipid Binding—Phosphatidylinositol phosphate strips (Invitrogen) were treated at room temperature for 5 h with 3% ovalbumin in TBS-T (10 mM Tris, pH 8.0, 150 mM NaCl, 0.1% Tween 20) to eliminate nonspecific binding, and incubated overnight at 4 °C with 1 g/ml talin fragments in TBS-T containing 3% ovalbumin. No of molecules in asymmetric unit Data set Wavelength (Å) Resolution (Å) Measured reflections Unique reflections Completeness (%) Rsym I/I
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