Figure 5.
The TLNRD1–CCM2 binding interface involves a hydrophobic groove on TLNRD1 and hydrophobic residues of CCM2. (A and B) Modeling of the TLNRD1–CCM2 complex using ColabFold using the TLNRD1 crystal structure (PDB accession no. 6XZ4) as a template. (A) Overall view of the predicted complex. The TLNRD1 monomers are colored blue, and the CCM2CTH helices are colored pink. The TLNRD1–CCM2 binding area is magnified, and the residues contributing to the interface are shown as sticks. (B) TLNRD1 has a hydrophobic channel (green) on the surface, which could facilitate CCM2 (pink) binding. The TLNRD1 four-helix module was colored by hydrophobicity using the AA index database (entry FASG890101 [Nakai et al., 1988]) in PyMOL, where green denotes hydrophobic residues and white polar residues. CCM2CTH is shown as sticks and predominantly contacts the hydrophobic region on TLNRD14H. (C) Comparison of the hydrophobic channel on the surface of TLNRD14H and the equivalent region on TLN1R8. The TLNRD12T mutant was designed to mimic the surface of TLN1R8. The green color denotes hydrophobic residues. On the TLNRD12E, the mutated basic residues are highlighted in blue. (D) Fluorescence polarization was used to determine the Kd of the interaction between TLNRD1 and various SUMO-CCM2CTH constructs (WT, I428S, I432D, and W412A/D422A). Kd values (nM) are shown in parentheses. ND, not determined. (E) Fluorescence polarization was used to determine the Kd of the interaction between CCM2CTH and various TLNRD14H constructs (WT, 2T, and 2E). Kd values (nM) are shown in parentheses. ND, not determined. (F) U2OS cells expressing various GFP-tagged CCM2 constructs and mito-TLNRD1-mScarlet or mito-mScarlet (CTRL) were imaged using a spinning disk confocal microscope. Representative single Z-planes are displayed. See also Fig. S5, E and F. Scale bars: (main) 25 µm and (inset) 5 µm. (G) U2OS cells expressing various GFP-tagged TLNRD1 constructs and mito-CCM2-mScarlet or mito-mScarlet (CTRL) were imaged using a spinning disk confocal microscope. Representative maximum intensity projections are displayed. Scale bars: (main) 25 µm and (inset) 5 µm.

The TLNRD1–CCM2 binding interface involves a hydrophobic groove on TLNRD1 and hydrophobic residues of CCM2. (A and B) Modeling of the TLNRD1–CCM2 complex using ColabFold using the TLNRD1 crystal structure (PDB accession no. 6XZ4) as a template. (A) Overall view of the predicted complex. The TLNRD1 monomers are colored blue, and the CCM2CTH helices are colored pink. The TLNRD1–CCM2 binding area is magnified, and the residues contributing to the interface are shown as sticks. (B) TLNRD1 has a hydrophobic channel (green) on the surface, which could facilitate CCM2 (pink) binding. The TLNRD1 four-helix module was colored by hydrophobicity using the AA index database (entry FASG890101 [Nakai et al., 1988]) in PyMOL, where green denotes hydrophobic residues and white polar residues. CCM2CTH is shown as sticks and predominantly contacts the hydrophobic region on TLNRD14H. (C) Comparison of the hydrophobic channel on the surface of TLNRD14H and the equivalent region on TLN1R8. The TLNRD12T mutant was designed to mimic the surface of TLN1R8. The green color denotes hydrophobic residues. On the TLNRD12E, the mutated basic residues are highlighted in blue. (D) Fluorescence polarization was used to determine the Kd of the interaction between TLNRD1 and various SUMO-CCM2CTH constructs (WT, I428S, I432D, and W412A/D422A). Kd values (nM) are shown in parentheses. ND, not determined. (E) Fluorescence polarization was used to determine the Kd of the interaction between CCM2CTH and various TLNRD14H constructs (WT, 2T, and 2E). Kd values (nM) are shown in parentheses. ND, not determined. (F) U2OS cells expressing various GFP-tagged CCM2 constructs and mito-TLNRD1-mScarlet or mito-mScarlet (CTRL) were imaged using a spinning disk confocal microscope. Representative single Z-planes are displayed. See also Fig. S5, E and F. Scale bars: (main) 25 µm and (inset) 5 µm. (G) U2OS cells expressing various GFP-tagged TLNRD1 constructs and mito-CCM2-mScarlet or mito-mScarlet (CTRL) were imaged using a spinning disk confocal microscope. Representative maximum intensity projections are displayed. Scale bars: (main) 25 µm and (inset) 5 µm.

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