Integrins are adhesion receptors that transmit force across the plasma membrane between extracellular ligands and the actin cytoskeleton. In activation of the transforming growth factor-β1 precursor (pro-TGF-β1), integrins bind to the prodomain, apply force, and release the TGF-β growth factor. However, we know little about how integrins bind macromolecular ligands in the extracellular matrix or transmit force to them. Here we show how integrin αVβ6 binds pro-TGF-β1 in an orientation biologically relevant for force-dependent release of TGF-β from latency. The conformation of the prodomain integrin-binding motif differs in the presence and absence of integrin binding; differences extend well outside the interface and illustrate how integrins can remodel extracellular matrix. Remodelled residues outside the interface stabilize the integrin-bound conformation, adopt a conformation similar to earlier-evolving family members, and show how macromolecular components outside the binding motif contribute to integrin recognition. Regions in and outside the highly interdigitated interface stabilize a specific integrin/pro-TGF-β orientation that defines the pathway through these macromolecules which actin-cytoskeleton-generated tensile force takes when applied through the integrin β-subunit. Simulations of force-dependent activation of TGF-β demonstrate evolutionary specializations for force application through the TGF-β prodomain and through the β- and not α-subunit of the integrin.
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This work was supported by National Institutes of Health grant R01AR067288, the Charles A. King Trust Postdoctoral Research Fellowship Program, Bank of America, N.A., Co-Trustee, and research collaboration with the Waters Corporation. For crystallography, SAXS, and simulations, we thank GM/CA-CAT beamline 23-ID at the Advanced Photon Source, beamline X9A at the National Synchotron Light Source, and the Pittsburgh Supercomputing Center, respectively.
The authors declare no competing financial interests.
Nature thanks T. Ha, Y. Shan and the other anonymous reviewer(s) for their contribution to the peer review of this work.
Extended data figures and tables
a, Superimposition of αVβ6 headpiece (Protein Data Bank accession number 4UM9, chains A and B)4 in closed conformation (αV yellow, β6 magenta) and head in open conformation in complex with pro-TGF-β1 (αV cyan, β6 green). b, c, Superimposed β3 and β6 βI domains, showing only moving regions in magenta or green (β6) and white (β3). d, Comparison of the macromolecular pro-TGF-β1 complex and soaked-in pro-TGF-β3 peptide complex showing integrin-binding loops (magenta and pink, respectively) and βI domain loops and metal ions (green and cyan, respectively). The conformation of the 215-RGDLATI-221 motif in intact pro-TGF-β1 when co-crystallized with αVβ6 is similar to that of the 241-RGDLGRL-247 motif in a pro-TGF-β3 peptide soaked into αVβ6 crystals4. e, The complete set of electron microscopy class averages (5,546 particles) from a gel filtration peak of the 2:2 pro-TGF-β1/αVβ6 complex. While most class averages show 2:2 complexes, 1:2 complexes and isolated αVβ6 are also present, presumably because of dissociation of 2:2 complexes. Scale bar in the first-class average is 50 Å.
a, Superimposition of bound and free pro-TGF-β1 prodomain monomers from the complex with αVβ6 and from an unbound (apo) porcine pro-TGF-β1 dimer3. b–d, The bowtie tail regions in pro-TGF-β1 crystal structures and the corresponding region of pro-BMP9, shown in identical orientations and vertically aligned after superposition on the arm domain. b, Integrin-bound human pro-TGF-β1 monomer. c, Bowtie tail regions in unbound monomers from the complex (orange), free human pro-TGF-β1 (B.Z., X.D. and T.A.S., unpublished observations, green), and free porcine pro-TGF-β1 (ref. 3) (yellow). Arg215 and Asp217 of the RGD motif in the unbound form are exposed to solvent but are not sufficiently accessible for integrin binding. d, BMP9 (ref. 13). e, Integrin-induced bowtie tail reshaping. Bowtie tails in the integrin-bound (cyan) and free (light blue) monomers are shown as backbone in thin stick with side chains participating in hydrogen bonds or interacting with the integrin or arm domain hydrophobic pocket shown as thick sticks. The side chains of residues N208 and T220 form hydrogen bonds to backbone in equivalent positions at a turn. Hydrogen bonds in the integrin-binding region and β9′-bridge region are shown in the same colour as sticks. The backbone shifts at residues 132–136 that line the bowtie groove. f, The integrin-bound bowtie tail is stabilized in a hydrophobic cleft of the arm domain. Bowtie tail backbone and side chains of key residues are shown as magenta sticks, and the remainder of the arm domain is shown as a silver surface with regions in close contact with the bowtie coloured violet-purple. Hydrogen bonds are shown as black dashed lines. Altogether, the HDX and analysis of crystal lattice contacts in Extended Data Figs 3 and 4 together with comparisons of monomer structures in Extended Data Fig. 2a–c provide insights into the flexibility of the bowtie tail and association regions. In the absence of integrin binding, the bowtie tail is dynamic. The structure of its C-terminal region is similar in the uncomplexed monomer here and a previous uncomplexed, porcine pro-TGF-β1 dimer (apo form)3. However, its N-terminal portion is either unstructured or dependent on crystal lattice contacts. The N-terminal association region is similarly either unstructured or adopts a structure that is dependent on crystal lattice contacts.
a, b, The pro-TGF-β1 moiety of the 2:1 complex (a) and isolated porcine pro-TGF-β1 (b) in identical orientations. Ribbon cartoons are coloured as in Fig. 1a. c, Unbound prodomain monomer from the complex structure (left) and prodomain monomer from isolated TGF-β1 (right) shown as ribbon cartoons with growth factor dimers in identical orientations. Ribbon cartoons are coloured according to the relative percentage of HDX at 10 s shown in the key, using peptides shown in Fig. 2a. d, e, Crystal lattice environments of integrin-bound human pro-TGF-β1 (d) and uncomplexed porcine pro-TGF-β1 (e). Portions of other molecules in the crystal that pack within 4 Å, including the bound integrin, are shown as white surfaces. Colouring in d is as in Fig. 2a and colouring in e is as in c, according to fast exchange rate as shown in the key. f, HDX-MS pepsin peptide coverage map of human pro-TGF-β1.
Extended Data Figure 4 Deuterium incorporation kinetics for all peptic peptides followed with HDX-MS.
Values represent the mean of three individual tests; error bars, s.d.
Data are merged from samples at 0.4 and 3 mg ml−1. a, Experimental SAXS of the 1:2 complex in solution (red, values represent mean and s.d of six datasets) versus the theoretical SAXS curve calculated with CRYSOL26 from the crystal structure of the 1:2 complex (cyan). b, Guinier analysis of the merged data shows a linear fit in the low q region. c, The Kratky plot exhibits a typical bell-shaped peak.
a–c, Surface plasmon resonance measurements of integrin αVβ6 headpiece binding to surface immobilized furin-mutant pro-TGF-β1 (a), endoglycosidase H (Endo H)-treated furin-mutant pro-TGF-β1 (b), and the untreated prodomain expressed in the absence of the growth factor (latency-associated peptide, LAP) (c). Curves are coloured red, 100 nM; dark green, 50 nM; blue, 20 nM; yellow, 10 nM; magenta, 5 nM; orange, 1 nM. The global fit to the 1:1 binding model is in black. Dissociation constant (Kd) and χ2 values from fits are shown. d, Binding in presence of Mg2+ of FITC-αVβ6 to wild-type or mutant pro-TGF-β1/GARP HEK293T co-transfectants as specific mean fluorescence intensity (percentage of wild-type). The mutated residues lie on the arm domain outside the RGDLATI motif. e, Activation of TGF-β1 by HEK293T cells co-transfected with pro-TGF-β1 and GARP, with or without αV and β6, assayed with luciferase reporter cells, and standardized with purified TGF-β1. The mutated residues lie on the arm domain outside the RGDLATI motif. f, Expression of wild-type and mutant pro-TGF-β1/GARP complexes on 293T transfectants determined by immunofluorescence flow cytometry with TW7-28G11 antibody. Values represent the mean and s.d. of three independent transfections in d–f. g, Hydrogen bonds within SDL2 in the β6 βI domain. h, The hydrogen bond network in the four residues, M224–P227, that link the bowtie tail to the β10 strand in the integrin-bound pro-TGF-β1 arm domain. Binding of integrin αVβ6 to pro-TGF-β1 does not liberate the growth factor3. The experiments in a–c address the question of whether integrin binding loosens the grip of the prodomain on the growth factor. Destabilization of the binding energy for the growth factor would require an identical stabilization of integrin binding to the isolated prodomain compared with the pro-complex; however, this is ruled out by the equivalent dissociation constants for integrin αVβ6 binding to the prodomain (7.6 nM, c) and pro-complex (8.2 nM, a). Thus, in the absence of force, there is no propagation of conformational change from the integrin binding site to the prodomain/GF interface that lowers affinity for ligand. The β6 SDL2 backbone is supported by many backbone hydrogen bonds (g), correlating with the finding that, in the open β6 conformation determined here, SDL2 is essentially identical in backbone to the previous closed conformation bound to the TGF-β3 peptide4. In contrast, SDL2 in β2 moves, even in intermediate headpiece opening17.
a–l, Force on the harmonic spring at the pulling end was measured every picometre in independent simulations at the indicated pulling rates. The four major events in straitjacket removal are arrowed in each simulation with two-letter codes. The first letter is F for fastener unsnapping and A for α2 helix unfolding. The second letter is I for the integrin-bound monomer and N for the non-bound monomer.
a–l, Force on the harmonic spring at the pulling end was measured every picometre in independent simulations at the indicated pulling rates. Pulling failed in each simulation (arrows) owing to thigh domain unfolding (thigh), β-propeller domain unfolding (β-prop) and separation of the αV β-propeller domain from pro-TGF-β1 and the β6 βI domain (αV unbinding).
Each simulation shows the beginning structure (rocking), the simulation (no rocking), and concludes with the final structure (rocking). αV and β6 simulations omitted the hybrid and thigh domains, respectively; these domains are added to videos to aid visualization of integrin orientation. (video 1) β6 pulling (Extended Data Fig. 7J). (videos 2-4) αV pulling that terminated with thigh domain unfolding (video 2, Extended Data Fig. 8d), β-propeller unfolding (video 3, Extended Data Fig. 8j), and αV unbinding (video 4, Extended Data Fig. 8l). (MP4 7942 kb)
Each simulation shows the beginning structure (rocking), the simulation (no rocking), and concludes with the final structure (rocking). αV and β6 simulations omitted the hybrid and thigh domains, respectively; these domains are added to videos to aid visualization of integrin orientation. (video 1) β6 pulling (Extended Data Fig. 7J). (videos 2-4) αV pulling that terminated with thigh domain unfolding (video 2, Extended Data Fig. 8d), β-propeller unfolding (video 3, Extended Data Fig. 8j), and αV unbinding (video 4, Extended Data Fig. 8l). (MP4 6423 kb)
Each simulation shows the beginning structure (rocking), the simulation (no rocking), and concludes with the final structure (rocking). αV and β6 simulations omitted the hybrid and thigh domains, respectively; these domains are added to videos to aid visualization of integrin orientation. (video 1) β6 pulling (Extended Data Fig. 7J). (videos 2-4) αV pulling that terminated with thigh domain unfolding (video 2, Extended Data Fig. 8d), β-propeller unfolding (video 3, Extended Data Fig. 8j), and αV unbinding (video 4, Extended Data Fig. 8l). (MP4 7947 kb)
Each simulation shows the beginning structure (rocking), the simulation (no rocking), and concludes with the final structure (rocking). αV and β6 simulations omitted the hybrid and thigh domains, respectively; these domains are added to videos to aid visualization of integrin orientation. (video 1) β6 pulling (Extended Data Fig. 7J). (videos 2-4) αV pulling that terminated with thigh domain unfolding (video 2, Extended Data Fig. 8d), β-propeller unfolding (video 3, Extended Data Fig. 8j), and αV unbinding (video 4, Extended Data Fig. 8l). (MP4 6271 kb)
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Dong, X., Zhao, B., Iacob, R. et al. Force interacts with macromolecular structure in activation of TGF-β. Nature 542, 55–59 (2017). https://doi.org/10.1038/nature21035
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