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Structural insights into integrin α₅β₁ opening by fibronectin ligand.

ABSTRACT: Integrin α₅β₁ is a major fibronectin receptor critical for cell migration. Upon complex formation, fibronectin and α₅β₁ undergo conformational changes. While this is key for cell-tissue connections, its mechanism is unknown. Here, we report cryo-electron microscopy structures of native human α₅β₁ with fibronectin to 3.1-angstrom resolution, and in its resting state to 4.6-angstrom resolution. The α₅β₁-fibronectin complex revealed simultaneous interactions at the arginine-glycine-aspartate loop, the synergy site, and a newly identified binding site proximal to adjacent to metal ion-dependent adhesion site, inducing the translocation of helix α1 to secure integrin opening. Resting α₅β₁ adopts an incompletely bent conformation, challenging the model of integrin sharp bending inhibiting ligand binding. Our biochemical and structural analyses showed that affinity of α₅β₁ for fibronectin is increased with manganese ions (Mn²⁺) while adopting the half-bent conformation, indicating that ligand-binding affinity does not depend on conformation, and α₅β₁ opening is induced by ligand-binding.

SUBMITTER: Schumacher S

PROVIDER: S-EPMC8104898 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Galectin-3 (Gal3) is a β-galactoside binding lectin that mediates many physiological functions, including the binding of cells to the extracellular matrix for which the glycoprotein α5β1 integrin is of critical importance. The mechanisms by which Gal3 interacts with membranes have not been widely explored to date due to the complexity of cell membranes and the difficulty of integrin reconstitution within model membranes. Herein, to study their interaction, Gal3 and α5β1 were purified, and the latter reconstituted into pore-suspended lipid bilayers comprised eggPC:eggPA. Using electrochemical impedance and fluorescence lifetime correlation spectroscopy, we found that on incubation with low nanomolar concentrations of wild-type Gal3, the membrane's admittance and fluidity, as well as integrin's lateral diffusivity, were enhanced. These effects were diminished in the following conditions: (i) absence of integrin, (ii) presence of lactose as a competitive inhibitor of glycan-Gal3 interaction, and (iii) use of a Gal3 mutant that lacked the N-terminal oligomerization domain (Gal3ΔNter). These findings indicated that WTGal3 oligomerized on α5β1 integrin in a glycan-dependent manner and that the N-terminal domain interacted directly with membranes in a way that is yet to be fully understood. At concentrations above 10 nM of WTGal3, membrane capacitance started to decrease and very slowly diffusing molecular species appeared, which indicated the formation of protein clusters made from WTGal3-α5β1 integrin assemblies. Overall, our study demonstrates the capacity of WTGal3 to oligomerize in a cargo protein-dependent manner at low nanomolar concentrations. Of note, these WTGal3 oligomers appeared to have membrane active properties that could only be revealed using our sensitive methods. At slightly higher WTGal3 concentrations, the capacity to generate lateral assemblies between cargo proteins was observed. In cells, this could lead to the construction of tubular endocytic pits according to the glycolipid-lectin (GL-Lect) hypothesis or to the formation of galectin lattices, depending on cargo glycoprotein stability at the membrane, the local Gal3 concentration, or plasma membrane intrinsic parameters. The study also demonstrates the utility of microcavity array-suspended lipid bilayers to address the biophysics of transmembrane proteins.

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Structural insights into integrin α₅β₁ opening by fibronectin ligand.

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OmicsDI is part of the ELIXIR infrastructure

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Dataset Information

Structural insights into integrin α5β1 opening by fibronectin ligand.

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OmicsDI is part of the ELIXIR infrastructure

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Structural insights into integrin α₅β₁ opening by fibronectin ligand.