Structural characterization of the multidomain talin–tensin3 interaction. (A) Representative images of NIH3T3 cells co-expressing GFP-R11DD-cBAK WT or carrying K2024E, K2031E, or both (K2024E+K2031E) with mCh-IDR. (B) Mitochondrial pulldown experiment using the same constructs as in A. (C) AlphaFold3 model of the talin R8–tensin3 TBS complex. TBS (orange) was predicted to engage the α2-α3 face of the R8 bundle (cyan). K1500 and R1510 (brown) are highlighted on the R11 domain. (D) NIH3T3 cells co-expressing GFP-R7R8-cBAK WT or carrying K1500E or R1510E with mCh-IDR. (E) Mitochondrial pulldown experiment using constructs as in D. (F) Cells co-expressing mCh-TNS3-WT-cBAK and GFP-TLN1 constructs (R8m, K1500E; R11m, K2024E+K2031E). mCh-TNS3-L702E-cBAK was used as a negative control. (G and H) Overlay 1H-15N HSQC spectra of 15N-labeled talin R3 (G) and talin R4 (H), at a concentration of 200 µM, in the absence (blue) and presence (red) of tensin3 TBS peptide at a molar ratio of 1:2. Magnified views in the right panels show the cross-peaks corresponding to the residues K869 and V871 of talin R3 (G) and the residues S927 and G969 of talin R4 (H), illustrating the progressive chemical shift changes at peptide molar ratios of 0 (blue), 0.25 (orange), 0.5 (green), 1.0 (coral), and 2.0 (red). The HSQC spectra of R3 and R4 were recorded at 700 and 800 MHz, respectively. (I and J) Mapping of the residue-specific CSD (related to Fig. S2, H and I) on the AlphaFold3 models of talin R3 (I) and R4 (J) colored in cyan, respectively, in complex with tensin3 TBS (orange). Residues with significant CSDs are colored red, using a red-white linear gradient scale with red corresponding to the maximum CSD and white to the threshold. Residues with CSDs below the threshold are colored in green. Images are generated using PyMOL. (K–M) ITC profiles of the talin R3 (K), R4 (L), and R7R8 (M) interaction with tensin3 TBS, respectively. The upper panels (and left panel in M) show the raw heat flow data obtained during the titration of 600 µM of tensin3 TBS peptide into 40 µM talin R3 (K) or R7R8 (M), or the titration of 450 µM of tensin3 TBS peptide into 30 µM talin R4 (L), respectively, at a temperature of 25°C. The lower panels (and right panel in M) represent the integrated heat per injection plotted against the molar ratio. The data were fitted using a single-site binding model with dissociation constants (Kd) of 20.5 ± 1.80 µM for the R3-TBS interaction (K), 23.8 ± 2.94 µM for the R4-TBS interaction (L), and 15.8 ± 0.90 µM for the R7R8-TBS interaction. All results are collected from three independent experiments. Scale bars (A, D, and F), 5 μm. CSD, chemical shift difference. Source data are available for this figure: SourceData F3.
Figure 3.

Structural characterization of the multidomain talin–tensin3 interaction. (A) Representative images of NIH3T3 cells co-expressing GFP-R11DD-cBAK WT or carrying K2024E, K2031E, or both (K2024E+K2031E) with mCh-IDR. (B) Mitochondrial pulldown experiment using the same constructs as in A. (C) AlphaFold3 model of the talin R8–tensin3 TBS complex. TBS (orange) was predicted to engage the α2-α3 face of the R8 bundle (cyan). K1500 and R1510 (brown) are highlighted on the R11 domain. (D) NIH3T3 cells co-expressing GFP-R7R8-cBAK WT or carrying K1500E or R1510E with mCh-IDR. (E) Mitochondrial pulldown experiment using constructs as in D. (F) Cells co-expressing mCh-TNS3-WT-cBAK and GFP-TLN1 constructs (R8m, K1500E; R11m, K2024E+K2031E). mCh-TNS3-L702E-cBAK was used as a negative control. (G and H) Overlay 1H-15N HSQC spectra of 15N-labeled talin R3 (G) and talin R4 (H), at a concentration of 200 µM, in the absence (blue) and presence (red) of tensin3 TBS peptide at a molar ratio of 1:2. Magnified views in the right panels show the cross-peaks corresponding to the residues K869 and V871 of talin R3 (G) and the residues S927 and G969 of talin R4 (H), illustrating the progressive chemical shift changes at peptide molar ratios of 0 (blue), 0.25 (orange), 0.5 (green), 1.0 (coral), and 2.0 (red). The HSQC spectra of R3 and R4 were recorded at 700 and 800 MHz, respectively. (I and J) Mapping of the residue-specific CSD (related to Fig. S2, H and I) on the AlphaFold3 models of talin R3 (I) and R4 (J) colored in cyan, respectively, in complex with tensin3 TBS (orange). Residues with significant CSDs are colored red, using a red-white linear gradient scale with red corresponding to the maximum CSD and white to the threshold. Residues with CSDs below the threshold are colored in green. Images are generated using PyMOL. (K–M) ITC profiles of the talin R3 (K), R4 (L), and R7R8 (M) interaction with tensin3 TBS, respectively. The upper panels (and left panel in M) show the raw heat flow data obtained during the titration of 600 µM of tensin3 TBS peptide into 40 µM talin R3 (K) or R7R8 (M), or the titration of 450 µM of tensin3 TBS peptide into 30 µM talin R4 (L), respectively, at a temperature of 25°C. The lower panels (and right panel in M) represent the integrated heat per injection plotted against the molar ratio. The data were fitted using a single-site binding model with dissociation constants (Kd) of 20.5 ± 1.80 µM for the R3-TBS interaction (K), 23.8 ± 2.94 µM for the R4-TBS interaction (L), and 15.8 ± 0.90 µM for the R7R8-TBS interaction. All results are collected from three independent experiments. Scale bars (A, D, and F), 5 μm. CSD, chemical shift difference. Source data are available for this figure: SourceData F3.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal