Figure 4. SHANK3 mutants show altered intra-domain conformational dynamics.

(A) C_α backbone RMSF calculated for the trajectories of the SHANK3^(1-346) fragment (PDB: 5G4X) either as WT or carrying one of the ASD-associated mutations R12C (blue) or L68P (red) show increased fluctuations for both mutants predominantly within the first 100 residues of the protein possibly suggesting a local mutation-induced increase in protein backbone dynamics. (B) C_α backbone RMSD determined for the full protein (aa 1–346). Within the first 100ns both mutants tend to show slightly increased backbone dynamics. This effect inverts with extended sampling time and mutants show on average reduced conformational dynamics compared to the WT. (C) C_α backbone RMSD restricted to the SPN domain of SHANK3 (aa 1–92). The traces resemble those observed for the full protein (aa 1–346) but absolute RMSD values are considerably larger for the SPN domain. (D) C_α backbone RMSD restricted to the ARR domain of SHANK3 (aa 93–346). The WT protein shows a stepwise increase in RMSD over time possibly indicating switches between distinct conformational states. This behavior is largely missing in both mutants but is more prominent for the L68P variant. (E) Local hotspots of conformational dynamics can be seen within the SPN domain (encircled) from overlays of individual frames of the trajectory (every fifth frame loaded, overlay [beginning:step:end]=0:20:20002 resulting in 1000 frames). RMSF = root mean square fluctuation, WT = wild type, ASD = autism spectrum disorders, RMSD = root mean square deviation.

Figure 4—video 1. MD trajectory of the SHANK3^(1-346) WT fragment.

Download video file ^{(16.7MB, mp4)}

To generate a visualization of peptide backbone motions in the SHANK3^(1-346) WT fragment, every hundredth frame of the 1000ns trajectory (10 ps/frame) analyzed in Figure 4 was used. Rotational and translational motion were removed and the trajectory was centered on the ARR domain. Individual images of the smoothed trajectory (smoothing window = 15) were rendered with POV-ray 3.7 and saved as video with 15 fps in Fiji. The conformational switch observed at 32–33 s in the video corresponds to the prominent RMSD increase observed approximately at 500ns (Figure 4B and C).

Figure 4—video 2. MD trajectory of the SHANK3^(1-346) R12C fragment.

Download video file ^{(15.2MB, mp4)}

The video was generated from the corresponding R12C trajectory as described for Figure 4—video 1. The conformational switch observed in the WT is absent in the R12C mutant. The mutated residue within the SPN domain is visualized with red spheres.

Figure 4—video 3. MD trajectory of the SHANK3^(1-346) L68P fragment.

Download video file ^{(16.9MB, mp4)}

The video was generated from the corresponding L68P trajectory as described for Figure 4—video 1. The conformational switch observed in the WT is also absent in the L68P mutant. The mutated residue within the SPN domain is visualized with red spheres.