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Structural basis for pure antagonism of integrin αVβ3 by a high-affinity form of fibronectin

Nature Structural & Molecular Biology volume 21, pages 383–388 (2014)Cite this article

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Abstract

Integrins are important therapeutic targets. However, current RGD-based anti-integrin drugs are also partial agonists, inducing conformational changes that trigger potentially fatal immune reactions and paradoxical cell adhesion. Here we describe the first crystal structure of αVβ3 bound to a physiologic ligand, the tenth type III RGD domain of wild-type fibronectin (wtFN10), or to a high-affinity mutant (hFN10) shown here to act as a pure antagonist. Comparison of these structures revealed a central π-π interaction between Trp1496 in the RGD-containing loop of hFN10 and Tyr122 of the β3 subunit that blocked conformational changes triggered by wtFN10 and trapped hFN10-bound αVβ3 in an inactive conformation. Removing the Trp1496 or Tyr122 side chains or reorienting Trp1496 away from Tyr122 converted hFN10 into a partial agonist. These findings offer new insights into the mechanism of integrin activation and a basis for the design of RGD-based pure antagonists.

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Figure 1: Binding properties of hFN10 and wtFN10 to αVβ3.
Figure 2: Structures of αVβ3 bound to FN10.
Figure 3: αVβ3–FN10 interfaces, conformational changes and structure validation.
Figure 4: RGD-containing loop structures in wild-type and modified FN10.

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Acknowledgements

We thank H.P. Erickson (Duke University) for providing the FN7-10 plasmid, T.J. Kunicki (The Scripps Research Institute) for access to AP5 antibody, M. Ginsberg (University of California, San Diego) for providing LIBS-1 and LIBS-6 mAbs, G.A. Petsko (Brandeis University) for helpful discussions and Z. Ding and D. Mueller-Pompalla for expert technical assistance. This work was supported by grants DK088327, DK48549, DK096334 and DK007540 (M.A.A.) from the National Institute of Diabetes and Digestive and Kidney Diseases, US National Institutes of Health.

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Author notes

  1. Johannes F Van Agthoven and Jian-Ping Xiong: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Medicine, Structural Biology Program, Harvard Medical School, Massachusetts General Hospital, Charlestown, Massachusetts, USA,

    Johannes F Van Agthoven, Jian-Ping Xiong, Brian D Adair & M Amin Arnaout

  2. Department of Medicine, Leukocyte Biology and Inflammation Program, Harvard Medical School, Massachusetts General Hospital, Charlestown, Massachusetts, USA,

    José Luis Alonso, Xianliang Rui & M Amin Arnaout

  3. Global Research and Early Development, Translational Innovation Platform, Oncology, Merck KGaA, Darmstadt, Germany

    Simon L Goodman

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Contributions

M.A.A. conceived and designed experiments. J.F.V.A., J.-P.X. and S.L.G. made and purified proteins. J.F.V.A. and J.-P.X. performed the crystallographic studies. J.L.A., X.R., J.F.V.A., M.A.A. and B.D.A. performed the biophysical, biochemical and cell-based assays. M.A.A., J.F.V.A., J.-P.X. and J.L.A. interpreted data. M.A.A. wrote the manuscript with the assistance of S.L.G. and J.-P.X.

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Correspondence to M Amin Arnaout.

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Integrated supplementary information

Supplementary Figure 1 Formation of αVβ3–FN10 complexes and fitted standard curve used to measure their Stokes radii.

(a) Coomassie-stained reduced SDS-PAGE gels of size-fractionated αVβ3 following its incubation with wFN10 (lanes 2, 5) or hFN10 (lanes 3, 6) in 1 mM each of Ca2+/Mg2+ or 1 mM Mn2+. 40 μg of protein mixtures were loaded in lanes 2, 3, 5, and 6. Lanes 1, 4 molecular mass markers (small arrows from top to bottom: 250, 150, 100, 75, 50, 37, 25, 20, 15, 10 kDa). αVβ3 formed a stable complex with hFN10 in Ca2+/Mg2+ or Mn2+ but with wtFN10 only in Mn2+. (b) Protein standards were Thyroglobulin (Thy), Ferritin (Fer), Albumin (Alb), Ovalbumin (Ova), chymotrypsinogen A (Chy), and ribonuclease A (Rib). Elution volumes were expressed as the square root of the log ratio of elution volume (Ve)/Void volume (Vo). Linear regression curve fit is plotted.

Supplementary Figure 2 Crystal structures and omit maps of αVβ3–FN10 complexes.

Ribbon diagrams of αVβ3 ectodomains bound to wtFN10 (a) or hFN10 (b). Both integrins are in the same orientation. αV chain is in blue and β3 chain is in light green (a) or pink (b). The four-αV domains (Propeller, Thigh, Calf-1 and Calf-2) and eight domains of β3 (PSI, βA, Hybrid [H], IE1-4 and βTD) are marked in (a). Orange spheres represent the five Mn2+ ions at the base of the Propeller and at the genu of αV. Mn2+ ions at LIMBS (gray), MIDAS (cyan) and ADMIDAS (magenta) are shown as spheres. Glycan carbons are indicated in the respective chain color. (c, d) σA weighted Fo-Fc omit electron density (ED) maps of the ligand-binding site and surrounding area for αVβ3-wtFN10 (c) and αVβ3-hFN10 (d) structures contoured at 4.0 σ, with FN10 protein omitted from the map calculation in each case.

Supplementary Figure 3 Significance of FN-glycan interaction in αVβ3–wtFN10 structure and IE2/Thigh and βTD/βA contacts found in the αVβ3–hFN10 structure.

(a) Adhesion (mean ± SD, n = 3 independent experiments) of HEK293T cells expressing αVβ3N339S or αVN266Qβ3N339S to immobilized full-length FN in presence of Ca2+/Mg2+. A540: absorbance at 540 nm. (b) Ribbon diagram showing the ionic and electrostatic interactions between β3's EGF-like domain 2 (IE2)(in pink) and αV's Thigh domain (in blue). σA weighted 2Fo-Fc map (gray, contoured at 1.0 σ) around the interacting residues is shown. The carboxyl oxygens of the β-genu residue Asp477 in β3-subunit IE2 H-bond to a carboxyl and carbonyl oxygens of Glu547 in the Thigh domain. OE1 and OE2 of the IE2 residue Glu500 H-bond to Glu550 OD1 and to Thr553 OG, respectively, with OE2 also forming a salt bridge with Lys503 of Thigh domain. (c) Ribbon diagram showing the intrachain H-bond between Ser674 in the CD loop of the βTD and Gln319 in the α6 helix of the βA domain. σA weighted 2Fo-Fc maps around Gln319 and the Asp672-Lys676 sequence is shown in gray contoured at 1.0 σ. α helices 1, 6 and 7 and strand F-α7 loop are labeled. The metal ions are colored as in supplementary Fig. 2.

Supplementary Figure 4 Crystal structure of αVβ3–hFN10/B complex.

Ribbon diagram of the αVβ3 ectodomain bound to hFN10/B (in light green). Integrin domains are labeled. Orange spheres represent the five Mn2+ ions at the base of the Propeller and at the αV genu. Glycan carbons are indicated in the respective chain color. Inset, σA weighted Fo-Fc omit map of the ligand-binding site and surrounding area for the αVβ3-hFN10/B structure contoured at 4.0 σ, with hFN10/B protein omitted from map calculation. Mn2+ ions at LIMBS, MIDAS and ADMIDAS are colored as in supplementary Fig. 2.

Supplementary Figure 5 Expression of the AP5 epitope on hFN10/B–bound αVβ3.

Histograms showing binding of fluoresceinated AP5 mAb to K562-αVβ3 cells in absence (control) or presence of wtFN10, hFN10 or hFN10/B in 1 mM MnCl2. Histograms show the results of two independent experiments (Exp).

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Van Agthoven, J., Xiong, JP., Alonso, J. et al. Structural basis for pure antagonism of integrin αVβ3 by a high-affinity form of fibronectin. Nat Struct Mol Biol 21, 383–388 (2014). https://doi.org/10.1038/nsmb.2797

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