Structural determination of the talin–tensin3 interaction. (A) SA composite omit map of tensin3 TBS electron density focused on chains C and D and contoured at 1σ (blue). (B) Refined 2F0-FC map of tensin3 TBS chain C and D contoured at 1σ (blue), with F0-FC map contoured at 3.5σ (+ve, green/−ve, red). (C) Electron density of the talin R8-tensin3 TBS complex (∼2.6 Å), with the refined 2F0-FC map of tensin3 TBS (orange) contoured at 0.7σ (blue). Talin R8, cyan. (D i and ii) AlphaFold3 model of the talin R11R12-tensin3 TBS complex (i) colored by confidence according to the pIDDT score with TBS facing forward, and contact prediction plot (ii) showing the expected positional error per residue in Angstrom (Å) ranging from 0 Å (dark green) to 30 Å (white). Note that the input talin R11R12 (cyan) and tensin3 TBS (orange) sequences are marked on the plot axes. (E) Poisson–Boltzmann electrostatic distribution map of the tensin3-binding surface of R11 in the predicted complex. Tensin3 peptide is shown in a stick representation with the hydrophobic residues labeled (red). The predicted R11-TBS complex is identical to the crystal structure shown in Fig. 2, A–C. (F i–iii) AlphaFold3 model of tensin3 TBS in complex with talin R3 (i), R4 (ii), and R7R8 (iii) with TBS facing forward. (G i–iii) Contact prediction plots related to F for the models of R3-TBS (i), R4-TBS (ii), and R7R8-TBS (iii). Note that the input talin (R3, R4, and R7R8; cyan) and tensin3 TBS (orange) sequences are marked on the plot axes. (H and I) Residue-specific CSD of talin R3 (H) and R4 (I) upon the addition of tensin3 TBS peptide at a 1:2 M ratio. The dashed line indicates the significant difference threshold of 5× SD. Mapping of CSD on the AlphaFold3 models is shown in Fig. 3, I and J. (J) Overlay 1H-15N HSQC spectra of 15N-labeled talin R7R8 (200 µM) in the absence (blue) and presence (red) of tensin3 TBS peptide at a 1:4 M ratio. Dashed boxes in the full spectra are magnified in the lower panels, illustrating the progressive chemical shift changes at peptide molar ratios of 0 (blue), 0.5 (orange), 1.0 (green), 2.0 (coral), and 4.0 (red). The HSQC spectra were recorded at 800 MHz. (K) Thermodynamic parameters of the talin R3–tensin3 TBS interaction. Red, blue, and green bars represent Gibbs free energy (ΔG = −26.8 ± 0.20 kJ/mol), enthalpy change (ΔH = 7.02 ± 0.62 kJ/mol), and entropy contribution (−TΔS = −33.8 ± 0.40 kJ/mol), respectively. (L) Thermodynamic parameters of the talin R4–tensin3 TBS interaction. ΔG = −26.3 ± 0.36 kJ/mol; ΔH = 12.3 ± 2.45 kJ/mol; -TΔS = −39.0 ± 2.62 kJ/mol. (M) Thermodynamic parameters of the talin R7R8–tensin3 TBS interaction. ΔG = −27.40 ± 0.17 kJ/mol; ΔH = 42.72 ± 2.06 kJ/mol; −TΔS = −70.10 ± 1.94 kJ/mol. Values in K‑M represent mean ± SD from triplicate measurements. CSD, chemical shift difference; SA, simulated annealing.
Figure S2.

Structural determination of the talin–tensin3 interaction. (A) SA composite omit map of tensin3 TBS electron density focused on chains C and D and contoured at 1σ (blue). (B) Refined 2F0-FC map of tensin3 TBS chain C and D contoured at 1σ (blue), with F0-FC map contoured at 3.5σ (+ve, green/−ve, red). (C) Electron density of the talin R8-tensin3 TBS complex (∼2.6 Å), with the refined 2F0-FC map of tensin3 TBS (orange) contoured at 0.7σ (blue). Talin R8, cyan. (D i and ii) AlphaFold3 model of the talin R11R12-tensin3 TBS complex (i) colored by confidence according to the pIDDT score with TBS facing forward, and contact prediction plot (ii) showing the expected positional error per residue in Angstrom (Å) ranging from 0 Å (dark green) to 30 Å (white). Note that the input talin R11R12 (cyan) and tensin3 TBS (orange) sequences are marked on the plot axes. (E) Poisson–Boltzmann electrostatic distribution map of the tensin3-binding surface of R11 in the predicted complex. Tensin3 peptide is shown in a stick representation with the hydrophobic residues labeled (red). The predicted R11-TBS complex is identical to the crystal structure shown in Fig. 2, A–C. (F i–iii) AlphaFold3 model of tensin3 TBS in complex with talin R3 (i), R4 (ii), and R7R8 (iii) with TBS facing forward. (G i–iii) Contact prediction plots related to F for the models of R3-TBS (i), R4-TBS (ii), and R7R8-TBS (iii). Note that the input talin (R3, R4, and R7R8; cyan) and tensin3 TBS (orange) sequences are marked on the plot axes. (H and I) Residue-specific CSD of talin R3 (H) and R4 (I) upon the addition of tensin3 TBS peptide at a 1:2 M ratio. The dashed line indicates the significant difference threshold of 5× SD. Mapping of CSD on the AlphaFold3 models is shown in Fig. 3, I and J. (J) Overlay 1H-15N HSQC spectra of 15N-labeled talin R7R8 (200 µM) in the absence (blue) and presence (red) of tensin3 TBS peptide at a 1:4 M ratio. Dashed boxes in the full spectra are magnified in the lower panels, illustrating the progressive chemical shift changes at peptide molar ratios of 0 (blue), 0.5 (orange), 1.0 (green), 2.0 (coral), and 4.0 (red). The HSQC spectra were recorded at 800 MHz. (K) Thermodynamic parameters of the talin R3–tensin3 TBS interaction. Red, blue, and green bars represent Gibbs free energy (ΔG = −26.8 ± 0.20 kJ/mol), enthalpy change (ΔH = 7.02 ± 0.62 kJ/mol), and entropy contribution (−TΔS = −33.8 ± 0.40 kJ/mol), respectively. (L) Thermodynamic parameters of the talin R4–tensin3 TBS interaction. ΔG = −26.3 ± 0.36 kJ/mol; ΔH = 12.3 ± 2.45 kJ/mol; -TΔS = −39.0 ± 2.62 kJ/mol. (M) Thermodynamic parameters of the talin R7R8–tensin3 TBS interaction. ΔG = −27.40 ± 0.17 kJ/mol; ΔH = 42.72 ± 2.06 kJ/mol; −TΔS = −70.10 ± 1.94 kJ/mol. Values in K‑M represent mean ± SD from triplicate measurements. CSD, chemical shift difference; SA, simulated annealing.

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