Fig. 4: Identification of Vemurafenib-binding sites in RIPK1 kinase.
From: Vemurafenib inhibits necroptosis in normal and pathological conditions as a RIPK1 antagonist
A The structural alignment of the initial (cyan cartoon) and final (yellow cartoon) conformations of Vemurafenib-bound RIPK1 during 100 ns MD simulations. The initial and last frames of Vemurafenib binding position were shown by red and green sticks. B Alignment of the initial (cyan cartoon) and final (yellow cartoon) conformations of the helix (residues 37–66), beta-strand (residues 10–36), and activation loop (residues 156–196) of Vemurafenib-bound RIPK1. Movement of helix, beta-strand, and activation loop is indicated by arrow. C Analysis of binding free energy contribution (MM/PBSA model) of key residues in the binding pocket. D Close-up view of the interaction between Vemurafenib and residue Leu157 of RIPK1. The orange stick structure represents Leu157 and the red stick represents Vemurafenib. The cyan cartoon represents the RIPK1 in the last frame of MD simulations. The yellow line indicates the possible electrostatic contacts between Leu157 of RIPK1 with Vemurafenib. E–G HEK293T cells were transfected with Flag-tagged WT, S161A mutant, L70A mutant, L157F mutant, and K45A mutant of RIPK1, respectively. After indicated treatment, the protein levels and phosphorylation of RIPK1 (E) were determined by western blot; the proteolysis rate of RIPK1 (F) was determined by DARTS Assay; the thermal stability of RIPK1 (G) was determined by CETSA assay. P value was calculated by unpaired Student’s t-test. (*p < 0.05, **p < 0.01, ***p < 0.001).
