Fig. 2. The RIPK3 kinase domain dimerizes using an interface similar to RAF.

(A) Superposition of RIPK3 (orange; amber; PDB ID: 4M66) and BRAF (4MNF; navy; sky blue) molecular dimers, illustrating conserved dimer surface. Inlay panel shows a magnified view of conserved arginine in molecular handshake across dimer interface. (B) Open-book representation of the RIPK3 dimer interface surface (amber). Residues selected for mutation to validate dimer interface are colored navy blue. Additional residues that contact the opposite kinase (<3 Å) are colored sky blue. Residue numbers are given in white. (C) RIPK3 dimer interface surface with one of the RIPK3 molecules depicted with space-filling model and the second RIPK3 molecule represented as orange ribbon diagram. (D) A close-up view of the RIPK3 dimer interface (PDB ID: 4M66). Residues mutated in this study are represented by sticks and labeled in black. Transparent sticks indicate additional points of contact, which were not mutated in this study. (E and F) Co-immunoprecipitation (IP) analysis of RIPK3 kinase domain (1 to 313 amino acids) interaction in lysates of HEK293T cells cotransfected with pcDNA3-Flag RIPK3 kinase domain and the indicated pcDNA3-HA-RIPK3 kinase domain that were immunoprecipitated for Flag. Blots are representative of at least three independent experiments. Normalized band intensities are means ± SD from all experiments. (G) Split luciferase complementation assay assessment of assessment of RIPK3 dimerization in HEK293T cells transfected with constructs expressing WT RIPK3 kinase domain fused to the C-terminal domain of click beetle luciferase (CBGC-RIPK3 KD) and the indicated RIPK3 kinase domain mutations fused to the N terminus of click beetle luciferase (CBGN-RIPK3 KD). Luciferase activity data are means ± SD combined from at least two independent experiments. **P < 0.01 and ***P < 0.005 by Mann-Whitney test. AU, arbitrary units.