Fig. 4: The N-terminal region of CD95 contributes to receptor aggregation.

A Superimposition of the two incomplete crystal structures (PDB:3THM in orange and PDB:3TJE in red). Using these structures, the complete amino-terminal region from R17 to G51 in PLAD was reconstituted by ab initio modeling and fused to the X-ray CD95 structures (in blue). Long molecular dynamics experiments (2µs) were finally performed to equilibrate the model. B The 3D organization of the CD95 dimeric model was generated by protein-protein docking of the protomer using SymmDock, with an imposed 2-fold symmetry. Most important amino acid involved in the interaction are depicted. C The 3D organization of the CD95 trimeric model was generated by protein-protein docking of the protomer using SymmDock, with an imposed 3-fold symmetry. D Indicated soluble CD95 constructs were expressed in HEK/293 T cells. E In denaturing and reducing conditions, the molecular weights of the soluble forms of full-length extracellular domain of CD95 (sCD95), Δ36 truncated counterpart (sCD95-Δ36) and K33G mutant (sCD95-K33G) were assessed by western blot. F In native condition, supernatants of the different sCD95-secreting HEK/293 T cells were resolved by gel filtration and, the quantity of sCD95 present in each fraction was measured by ELISA. The molecular weights of full-length extracellular domain of CD95 (sCD95) and sCD95-Δ36 constructs were assessed using native molecular markers. For each fraction, the percentage of dosed CD95 was depicted. G In native condition, the molecular weights of full-length extracellular domain of CD95 (sCD95) (also depicted in F) and sCD95-K33G constructs were assessed by gel filtration as described in (F).