Structural determinants of integrin β-subunit specificity for latent TGF-β

Abstract

Eight integrin α-β heterodimers recognize ligands with an Arg-Gly-Asp (RGD) motif. However, the structural mechanism by which integrins differentiate among extracellular proteins with RGD motifs is not understood. Here, crystal structures, mutations and peptide-affinity measurements show that αVβ6 binds with high affinity to a RGDLXXL/I motif within the prodomains of TGF-β1 and TGF-β3. The LXXL/I motif forms an amphipathic α-helix that binds in a hydrophobic pocket in the β6 subunit. Elucidation of the basis for ligand binding specificity by the integrin β subunit reveals contributions by three different βI-domain loops, which we designate specificity-determining loops (SDLs) 1, 2 and 3. Variation in a pair of single key residues in SDL1 and SDL3 correlates with the variation of the entire β subunit in integrin evolution, thus suggesting a paradigmatic role in overall β-subunit function.

Figure 1

Activation and binding of pro-TGF-β1 by wild-type and mutant α_V integrins. (a) Indicated HEK293T transfectants assayed for TGF-β1 activation with mink lung luciferase reporter cells, measured as relative light units (RLU). Mock, control mock transfection. (b) Saturation binding of FITC–pro-TGF-β1 to HEK293T transfectants, shown as percentage mean fluorescence intensity (% MFI) of α_V P2W7 monoclonal antibody binding. Slashes denote ‘and.’ (c,d) Binding of peptides to α_Vβ₆ (c) or α_Vβ₃ (d) headpieces, measured by fluorescence anisotropy. Anisotropy is measured as millianisotropy units (mA), as (F_‖ – F_⊥)/(F_‖ + 2F_⊥) × 1,000, where F_‖ is the fluorescence intensity parallel to the excitation plane, and F_⊥ is the fluorescence intensity perpendicular to the excitation plane. Data show mean ± s.e.m. of technical triplicate samples. Peptides at the indicated concentrations were used with 200 nM α_Vβ₆ or 4 µM α_Vβ₃ headpiece and 5 nM of fluorescent peptide probe. K_d was calculated from IC₅₀ as described.

Figure 2

Crystal structures and comparisons of the α_Vβ₆ headpiece. (a) Overall ribbon diagram of the α_Vβ₆ headpiece (with each domain in a different color) with pro-TGF-β3 peptide (magenta). (b) Conformational change of the βI α2-α3 loop in the absence and presence of pro-TGF-β3. Carbon color code: green, in absence of peptide α_V; cyan, β₆; yellow, in presence of peptide β₆; magenta, peptide aspartate. Metals are white or gray spheres. (c) pH dependence of binding affinity. Binding of FITC–pro-TGF-β3 peptide measured with fluorescence anisotropy is shown. Data show mean ± s.e.m. of technical triplicate samples. (d,e) Ligand binding of α_Vβ₆ to pro-TGF-β3 peptide (d) and α_Vβ₃ to cilengitide (e). Carbon color code: green, α_V; cyan, β₃ or β₆, with different shades for SDLs 1, 2 and 3; magenta, ligands. The MIDAS metal ion is a silver sphere. (f,g) Key residues that contribute to packing between SDLs 1, 2 and 3 in β₆ (f) and β₃ (g). SDL color code is as in d and e. Van der Waals surfaces around interacting side chains are shown as dots. (h) Phylogenetic tree for integrin β-subunit SDL sequences. Ligand-contacting residues in SDL1 and SDL3 in the X₁ positions are highlighted in pink. Residues that form packing interactions of SDL1 and SDL3 with SDL2 in the X₂ position are highlighted in orange. Cysteines forming disulfides are highlighted in yellow. Residues that coordinate metals are asterisked in orange (MIDAS), green (ADMIDAS) and cyan (SyMBS).

Figure 3

Ligands of α_Vβ₆. (a) RGD sequences from pro-TGF-β and VP1 protein from FMDV. (b) Competitive binding affinities of TGF-β3 peptide truncations. Fluorescence anisotropy data are mean ± s.e.m. of technical triplicate samples scaled logarithmically. (c) Western blot of pro-TGF-β1 secreted by the indicated HEK293T transfectants, with antibody to the prodomain as described previously. (d) TGF-β bioassay of pro-TGF-β1 and its double-proline mutant. Data show mean ± s.e.m. of technical triplicate samples.