Structure-based mutations in tensin3 disrupt its interaction with talin. (A) Crystal structure of the talin R11R12–tensin3 TBS (aa 692–718) complex. (i) TBS (orange) forms a six-helix bundle with R11 (cyan). (ii) TBS engages the α2-α5 face of R11. (B) R11-TBS interface is stabilized by multiple electrostatic and hydrophobic interactions. K2024, K2119, and K2133 (black), form electrostatic contacts with D696, S698, and D710 (red). (C) Poisson–Boltzmann electrostatic distribution map of the tensin3-binding surface of R11. Tensin3 peptide is shown in sticky representation with the hydrophobic residues labeled (red). (D) GFP-TLN1-cBAK was co-expressed with mCh-IDR wild-type (WT), deletion of TBS (ΔTBS), or those carrying the point mutation L702E, I706E, or L707E, respectively, in NIH3T3 cells. Groups of GFP-cBAK and mCh-IDR-ΔTBS were used as negative controls. (E) Summary table of D. (F) Mitochondrial pulldown experiment with the constructs used in D. (G) Quantification of F from triplicate experiments. Data are normalized to WT. Error bars are SEM; ** indicates P < 0.01 (ordinary one-way ANOVA with Dunnett’s multiple comparisons). (H) Representative images of NIH3T3 cells expressing mCh-TNS3-WT-cBAK or mCh-TNS3-L702E-cBAK and GFP-TLN1 (left panel) or GFP-TLN2 (right panel). mCh-cBAK was used as a negative control to recruit GFP-TLN1 and GFP-TLN2 to the mitochondria. Data are collected from three independent experiments. Scale bars (D and H), 5 μm. Source data are available for this figure: SourceData F2.
Figure 2.

Structure-based mutations in tensin3 disrupt its interaction with talin. (A) Crystal structure of the talin R11R12–tensin3 TBS (aa 692–718) complex. (i) TBS (orange) forms a six-helix bundle with R11 (cyan). (ii) TBS engages the α2-α5 face of R11. (B) R11-TBS interface is stabilized by multiple electrostatic and hydrophobic interactions. K2024, K2119, and K2133 (black), form electrostatic contacts with D696, S698, and D710 (red). (C) Poisson–Boltzmann electrostatic distribution map of the tensin3-binding surface of R11. Tensin3 peptide is shown in sticky representation with the hydrophobic residues labeled (red). (D) GFP-TLN1-cBAK was co-expressed with mCh-IDR wild-type (WT), deletion of TBS (ΔTBS), or those carrying the point mutation L702E, I706E, or L707E, respectively, in NIH3T3 cells. Groups of GFP-cBAK and mCh-IDR-ΔTBS were used as negative controls. (E) Summary table of D. (F) Mitochondrial pulldown experiment with the constructs used in D. (G) Quantification of F from triplicate experiments. Data are normalized to WT. Error bars are SEM; ** indicates P < 0.01 (ordinary one-way ANOVA with Dunnett’s multiple comparisons). (H) Representative images of NIH3T3 cells expressing mCh-TNS3-WT-cBAK or mCh-TNS3-L702E-cBAK and GFP-TLN1 (left panel) or GFP-TLN2 (right panel). mCh-cBAK was used as a negative control to recruit GFP-TLN1 and GFP-TLN2 to the mitochondria. Data are collected from three independent experiments. Scale bars (D and H), 5 μm. Source data are available for this figure: SourceData F2.

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