Structural insights into integrin α5β1 opening by fibronectin ligand

See allHide authors and affiliations

Science Advances  07 May 2021:
Vol. 7, no. 19, eabe9716
DOI: 10.1126/sciadv.abe9716
  • Fig. 1 Cryo-EM analysis of FN-bound integrin α5β1 reveals an open conformation.

    (A) Two views of the FN7-10 (orange)–bound α5β1 (green, α subunit; blue, β subunit) cryo-EM structure, stabilized by TS2/16 (pink). The headpiece reveals an open conformation. (B) Schematic of integrin α5β1 domain architecture and nomenclature of different parts (headpiece, head, upper, and lower legs), including FN7-10 and TS2/16. TM, transmembrane; PM, plasma membrane; CT, cytoplasmic tail. (C) Molecular model based on the cryo-EM density, showing the same two views as (A). (D) Close-up and overlay of cryo-EM density with the modeled synergistic metal ion–binding site (SyMBS or LIMBS), MIDAS, and ADMIDAS within the βI domain.

  • Fig. 2 Binding of integrin α5β1 to FN7-10 occurs through an extensive interface, including RGD, FN synergy site, and juxta-ADMIDAS area.

    (A) Overview of the α5β1-FN interface. (B) The RGD loop of FN10 inserted into the pocket between α5 and β1 subunits. It binds to Q221, D227(α5), and the MIDAS cation in β1. Newly found contacts are made between P1497(FN10)-Y133(β1) and P1497(FN10)-S134(β1). A, ADMIDAS; M, MIDAS; S, SyMBS (LIMBS). (C) Key interactions in the ADMIDAS region: salt bridge R1445(FN10)-E320(β1); hydrogen bond Y1446(FN10)-D137(β1). (D) The synergy site of FN9 contacts α5: salt bridge R1379(FN9)-D154(α5); interaction of P1376(FN9)-Y208(α5). R1374 is not visibly engaged. (E) Arrangement of secondary structure elements around ADMIDAS and FN. (F) Solid-phase binding assays show the affinity of FN7-10 to integrin ectodomain mutants. Left: Binding of ND-α5β1 to FN7-10 mutants in the presence of Mn2+ (normalized to FN7-10 wild type). Middle: Binding of recombinant, unclasped α5β1 ectodomain mutants to FN7-10 in the presence of Mn2+ (normalized to wild-type ectodomain). Bars represent means + SD of three measurements. Right: Assay strategy. a.u., arbitrary units; wt, wild type. (G) Hydrophobic interactions between the antibody TS2/16 and β1 centered around K208(β1). (H) Close-up of the cation-π interaction between K208(β1) and Y100(TS2/16 CDR-H3) overlaid with cryo-EM density (gray).

  • Fig. 3 N-glycosylation of integrin α5β1 bridges interface to FN.

    (A) Table listing the N-glycosylation sites and the types of glycan trees as determined in our structure. (B) Electrostatic potential map of the FN-integrin interface and the N275-glycan. (C) Two views of the N275-glycan on α5, which is part of the extensive α5β1-FN interface. The N275-glycan contacts with FN9. (D) Comparison of glycan position between integrin α5β1 and integrin αvβ3 [PDB ID: 3ije (43)] and αvβ3 in complex with FN10 [PDB ID: 4mmz (42)]. The N275-glycan (α5β1) is a conserved glycosylation site among FN-recognizing integrins such as αvβ3. (E) Solid-phase binding assay to measure the binding affinity of integrin α5(N275A)β1 ectodomain mutant in comparison to wild-type ectodomain. Both wild-type and N275A ectodomain mutant were unclasped and binding to FN7-10 wild type evaluated in the presence of Mn2+. Bars represent means + SD of three measurements. Unspecific binding of integrin to bovine serum albumin (BSA)–coated control wells was subtracted from the total absorbance values. Normalized binding is expressed as the ratio of mutant absorbance relative to wild-type absorbance (ratio = 1 represents wild-type binding).

  • Fig. 4 Resting integrin α5β1 reveals an incompletely bent conformation.

    (A to C) Three views of the resting integrin α5β1 cryo-EM structure. The position of the membrane is guided in yellow. Note that precise direction of the membrane cannot be determined because of the lack of membrane density from the structure. (D to F) The structure of the headpieces and modeled structure using homology models of PDB ID: 3fcs (13) for the lower legs. The headpiece reveals a closed conformation, with an orthogonal angle between the headpiece and legs. (G) The lower legs of integrin α5β1 are parallelly contacting each other, between EGF1/2 (β1) and calf1 (α5). (H) Multibody analysis revealed flexibility between the headpiece and lower legs, with an angle varying from 71° to 93° between the first analyzed state (“frame 1”) and the last state (“frame 10”). See fig. S7 for all the frames.

  • Fig. 5 Resting integrin α5β1 is disadvantageous for FN binding.

    (A) An overlay of the βI, hybrid, and PSI domains of resting (blue) and FN-bound integrin (gray) shows the movement of the α1 and α7 helices in the βI domain and the subsequent swing-out motion of the hybrid domain. (B) ADMIDAS is farther from FN10 (Y1446) in the resting βI domain (blue) than in the open βI domain (gray). (C) Comparison of the position of glycanN343 on β1 with (top) and without (bottom) ligand binding. The glycanN343 hinders the FN binding in the resting structure.

  • Fig. 6 FN-binding of NDs containing α5β1 is enhanced by Mn2+.

    (A) Cryo-EM image of α5β1 with various concentrations of Mn2+. 2D class averages are shown, revealing no integrin opening. (B) Solid-phase binding assays to measure integrin binding to full-length FN (FN-FL) in the presence of activating (Mn2+) or resting (Ca2+/Mg2+) buffer conditions and varying integrin α5β1 concentration. Three individual experiments were conducted, and all conditions were measured and averaged (points represent means + SD). Control measurements of α5β1 on BSA are shown.

  • Fig. 7 Integrin α5β1 exists in an equilibrium of conformational states that can be altered by a combination of ligands FN7-10.

    (A) FN7-10 binding to α5β1 was tested by SEC under resting (Ca2+/Mg2+) and activating (Mn2+) buffer conditions. In the presence of Mn2+, FN7-10 binding is detected by SDS-PAGE. The peak containing ND-α5β1 and FN7-10 is marked with asterisk. MSP, membrane scaffolding protein used to form NDs. The positions of molecular weight standards for gel filtration are marked with arrowheads on the chromatograms. (B) The effect of the antibody TS2/16 on α5β1 binding to FN7-10 was tested in the presence or absence of MnCl2. When Mn2+, FN7-10 and TS2/16 are mixed together with ND-α5β1, a peak shift can be observed (#). The positions of molecular weight standards for gel filtration are marked with arrowheads on the chromatograms. (C) Summary of the negative staining quantification of ND-α5β1 alone or mixed with FN7-10 and TS2/16 under resting (Ca2+/Mg2+) and activating (Mn2+) buffer conditions. Particles were sorted into three states: “bent,” “intermediate,” and “separated legs.” For each condition, ~20,000 particles were initially picked, and those displaying a clear signal (i.e., ~5000 to 8000) were selected for the final quantification. Examples of 2D classes are shown.

  • Fig. 8 Model of integrin α5β1 opening.

    Integrin α5β1 exists in an equilibrium of conformational states, fluctuating between bending and extension. FN binding secures the extended and open conformation of integrin α5β1 through three binding sites (marked as 1, 2, and 3) at the integrin heads. Mn2+ primes binding sites, SyMBS (LIMBS), ADMIDAS, and MIDAS and brings integrin to a ready state in which it is ready to bind to FN. Full engagement of FN takes place, resulting in the integrin α5β1 opening. Note that lower leg opening was not visible in our integrin α5β1 structure and, therefore, the opened lower leg is based on previous biochemical observation (15).

Supplementary Materials

  • Supplementary Materials

    Structural insights into integrin α5β1 opening by fibronectin ligand

    Stephanie Schumacher, Dirk Dedden, Roberto Vazquez Nunez, Kyoko Matoba, Junichi Takagi, Christian Biertümpfel, Naoko Mizuno

    Download Supplement

    The PDF file includes:

    • Table S1
    • Figs. S1 to S8

    Other Supplementary Material for this manuscript includes the following:

    Files in this Data Supplement:

Stay Connected to Science Advances

Navigate This Article