Insights into the membrane repair mechanism by the coiled-coil-mediated oligomerization of TRIM72

Figures

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  Fig. 1. | Structure of TRIM72. A. Domain organization of TRIM72. Each domain is depicted as follows: RING (green), B-box (orange), H1 (magenta), H2 (cyan), L2 (teal), H3 (yellow), and PRY-SPRY (white). Residues and small motifs are annotated on the top and bottom, respectively. B. Overall structure of TRIM72 represented by a surface and ribbon diagram. The color of each domain corresponds to the domain architecture in panel (A). The second protomer is labeled with a prime symbol (′) and depicted in less vibrant colors. PRY-SPRY domains are presented with an electrostatic potential surface (positive and negative charges are indicated in blue and red, respectively). Positively charged residues are represented as a stick model. The proposed membrane-binding mode of TRIM72 is also presented. C. Buried surface area of a protomer, illustrated in yellow color, measuring approximately 5000 Å2. For clarity, the other promoter is presented as a ribbon diagram.

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  Fig. 2. | Flexibility of the RBCC domain of TRIM72. A.Ab initio DAMMIN bead models of full-length TRIM72 WT (upper) and TRIM72 ΔRING (lower). Ten bead models of each protein are superimposed. B. Small-angle X-ray scattering molecular envelope models of TRIM72 (upper) and TRIM72 ΔRING (lower). High-resolution crystal structures of the TRIM72 ΔRING dimer (magenta) and RING (green) are superimposed and presented as ribbon diagrams. Each envelope model was generated from an average of 10 bead models in panel (A). The additional volume at both ends of the full-length TRIM72 is expected to represent the RING domain in its position when in an open conformation in solution. C. Conformational movement of the coiled-coil. Superposition of all eight determined structures reveals that the core formed by the central H1:H1′ and H3:H3′ helices and a pair of PRY-SPRY domains is highly rigid. In contrast, the peripheral H1 and H2 helices and B-box domain are highly flexible with a maximum movement of 20 Å. Notably, the coiled-coil moves in only one direction, moving closer to and farther from the membrane. D. Plot of the RMSD of equivalent Cα atoms between full-length and various models as a function of…

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  Fig. 3. | Molecular dynamics simulation of TRIM72. A. Solute potential energy over a 30-ns trajectory. B. Domain-specific difference of the root-mean-square fluctuations (RMSF) of TRIM72 during the 30-ns trajectory. The residues of the coiled-coil domain used for calculating RMSF values along the Cartesian axis are highlighted in transparent green. C. “Up-and-down” movement of TRIM72 at the different time points during the MD simulation. Models are extracted at 0 ns (yellow), 10 ns (magenta), 20 ns (orange), and 30 ns (aquamarine) for clarity. The ribbon diagrams are presented from the side (top panel) and top views (bottom panel). D. RMSF calculation of the coiled-coil domain along the Cartesian axis. Each axis is illustrated in panel (C). E. Dynamic interactions between the dimeric heptad and the L2 linker. Ribbon diagrams (left) and buried surface area plots (right) for each model in the MD simulation trajectory are depicted in panel (C).

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  Fig. 4. | Higher-order TRIM72 assembly on the lipid membrane. A. Cryo-electron micrograph showing proteoliposomes with the TRIM72 oligomer on phosphatidylserine-SUVs. TRIM72-TRIM72 contacts were observed in the peripheral region between separate liposomes (left). Black scale bars indicate 20 nm. Box plot of the distance between the TRIM72 oligomer-bound liposomes (right). Mean and median values (small and long bars in the box, respectively) display the first and third quartiles (upper and lower ends of the box, respectively). Each value (dot) is represented with whiskers from minimum to maximum. B. The molecular model of vesicle-patching assembly. Top view (left) and side view (middle) rotated by 90°. The model is derived from the crystal packing of TRIM72 WT (7XYY). Notably, the distance between the patched liposomes is approximately 10 nm on the cryo-EM image, which is close to the height of the oligomer at approximately 11 nm in the middle of panel (B). The dashed lines indicate two sides of the membrane surface.