Fig. 5: N545 is relevant for hRIPK1 nucleation and heteromerization. | Nature Communications

Fig. 5: N545 is relevant for hRIPK1 nucleation and heteromerization.

From: Structural basis for amyloid fibril assembly by the master cell-signaling regulator receptor-interacting protein kinase 1

Fig. 5

A 1H-15N HSQC overlay of ~8 µM RIPK1 WT (magenta) and ~21 µM RIPK1 N545D (blue) RHIM constructs (left) in 20 mM MES, pH 6.6, and traces corresponding to the first increment of this HSQC spectra (labeled with t0, magenta for WT, navy blue for N545D), after one hour (t1h, purple for WT, cyan for N545D), and after overnight incubation (to/n, orange for WT, teal for N545D, with o/n standing for overnight), highlighting the difference in intensity decay between WT and mutant. Experiments were collected on an 800 MHz spectrometer (1H frequency). B Bicistronic coexpression of His-tagged RIPK1 (WT or N545D) and TwinStrep-tagged RIPK3. Solubilized material was purified by His-affinity chromatography, and eluates were analyzed by Western blot using anti-His (left) and anti-Strep antibodies (right). RIPK3 co-purified robustly with WT RIPK1 (labeled as “WT”) but was barely detectable with RIPK1 N545D (labeled as “Mut”, from “Mutant”), indicating that N545 is essential for RIPK1–RIPK3 heteromeric fibril association. A control lane containing a 1:1 mixture of individually expressed monomeric His-tagged-RIPK1 and TwinStrep-tagged-RIPK3 is shown, labeled as “C”, from “Control”. This experiment was repeated five times with similar results. Source data are provided as a Source Data file.

Back to article page