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Mechanistic basis for the recognition of laminin-511 by α6β1 integrin

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Science Advances  01 Sep 2017:
Vol. 3, no. 9, e1701497
DOI: 10.1126/sciadv.1701497
  • Fig. 1 The integrin binding region of LM511.

    (A) Front (left) and lateral (right) faces of the integrin binding region of LM511 that consists of chains α5 (green), β1 (blue), and γ1 (red). LG1–3 of the α5 chain are colored in deep green (LG1), green-cyan (LG2), and yellow-green (LG3). A calcium ion at the LG1-LG3 interface is shown as a magenta sphere, disulfide-linked Cys residues as yellow sticks, and Cα atoms of C-terminal residues as orange spheres. (B) Octahedral calcium coordination in the LG1-LG3 interface. (C) Triangular cloverleaf configuration of LG1–3. (D) Schematic illustration of the configurational change of LG1–3 from “open” to “cloverleaf” through coiled-coil assembly.

  • Fig. 2 Association of LG1–3 with β1-γ1 dimer.

    (A) Back view showing LG1-LG2 (surfaces), β1-γ1 dimer (ribbons) with side chains (sticks), and water molecules (spheres). (B) Hydrophobic patch on LG1. (C) Hydrophobic interactions between the hydrophobic patch of LG1 and the C-terminal region of the β1-γ1 dimer. (D) The interaction between LG2 and β1 chain was mediated by a layer of hydrogen-bonded water molecules. 2Fo-Fc electron density map countered at 1.0σ is shown as blue mesh around water molecules.

  • Fig. 3 Close contact of the γ1-tail with the headpiece of α6β1 integrin.

    (A) Electron microscopic image of LM511E8 complexed with α6β1 integrin (see also fig. S3). (B) Schematic illustration of the interaction between LM511E8 and α6β1 integrin. (C) Amino acid sequences of wild-type (WT) and Cys-substituted γ1-tails. Cys-substituted residues are shown in yellow. (D) The residues cross-linked to the γ1-tail (yellow sticks) lie near the metal ion (green sphere) in the MIDAS site of integrin β1 (β1-MIDAS) (Protein Data Bank ID: 4WJK). The β1-MIDAS metal ion and water molecules are depicted as green and red spheres, respectively. (E) Intermolecular disulfide formation between LM511E8 having γ1-I1606C (top) or γ1-K1608C (bottom) substitution and Cys-substituted α6β1 integrins (see also fig. S6). Arrowheads indicate disulfide-linked products. (F) Distinct topologies of the γ-tail (top) and the RGD motif (bottom) on the integrin’s headpiece.

  • Fig. 4 Direct contribution of γ1E1607 to laminin-integrin interaction.

    (A) Inhibition of the LM511E8–α6β1 integrin interaction by γ1-tail–derived peptides [white, γ1C5; black, γ1C5(EQ)] in the absence (circle) or presence (square) of the integrin β1 activating monoclonal antibody (mAb) TS2/16. IC50 values of peptides (means ± SD of three independent experiments) are shown in the table (right). (B) Schematic model for the mechanism by which integrin recognizes laminin.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/3/9/e1701497/DC1

    fig. S1. Preparation and crystallization of tLM511E8.

    fig. S2. Comparison of the crystal structures of tLM511E8 and mini-E8 of LM111.

    fig. S3. Electron microscopic imaging of the LM511E8–α6β1 integrin complex.

    fig. S4. Cys-substituted residues on βI domain.

    fig. S5. Integrin binding activity of wild-type and Cys-substituted LM511E8s.

    fig. S6. Disulfide formation between Cys-substituted LM511E8 and α6β1 integrin.

    fig. S7. Disulfide cross-link assays using LM511E8/I1606C/EQ and LM511E8/K1608C/EQ.

    fig. S8. Inhibition of the LM511E8–α6β1 integrin interaction by wild-type and Δγ1C5 LM511E8.

    table S1. Data collection and refinement statistics.

  • Supplementary Materials

    This PDF file includes:

    • fig. S1. Preparation and crystallization of tLM511E8.
    • fig. S2. Comparison of the crystal structures of tLM511E8 and mini-E8 of LM111.
    • fig. S3. Electron microscopic imaging of the LM511E8–α6β1 integrin complex.
    • fig. S4. Cys-substituted residues on βI domain.
    • fig. S5. Integrin binding activity of wild-type and Cys-substituted LM511E8s.
    • fig. S6. Disulfide formation between Cys-substituted LM511E8 and α6β1 integrin.
    • fig. S7. Disulfide cross-link assays using LM511E8/I1606C/EQ and LM511E8/K1608C/EQ.
    • fig. S8. Inhibition of the LM511E8–α6β1 integrin interaction by wild-type andΔγ1C5 LM511E8.
    • table S1. Data collection and refinement statistics.

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