Comparative evolutionary analysis of RIPK1-5. (A) Domain structures of human RIP kinases. RHIM = RIP homotypic interaction motif, DD = death domain, CARD = caspase activation and recruitment domain, ANK = ankyrin repeats. (B) Positive selection analysis of RIPK1-5 in the indicated mammalian order. Input sequences and PAML p-values can be found in Supplementary Files 1 and 2. Images of model species generated using BioRender. (C) Heat amp showing the percentage of species within a clade that have the indicated protein. The clades and the number of species within each clade are indicated on the left. Complete lists of proteins and species in each group can be found in Supplementary Files 4 and 5.

Diverse vertebrate RIPK3 proteins activate NF-κB. (A) Percent similarity of RIPK3 from the indicated species compared to humans. (B) RIPK3 proteins were transfected into WT or RIPK1 KO HEK293T cells, along with NF-κB firefly luciferase and control renilla luciferase reporter plasmids (see Materials and Methods), and NF-κB activity was measured at 18h post-transfection. (C) RIPK3 proteins were transfected into HEK293T cells with and without human ZBP1 and MLKL. At 18h post-transfection, cells were stained using the ReadyProbe Cell Viability kit and fluorescence was measured using a plate reader. Species shown are mouse (Mus musculus), cat (Felis catus), pig (Sus scrofa), lizard (Anolis carolinensis), turtle (Chelonia mydas), and lamprey (Petromyzon marinus). Data are representative of 3-5 independent experiments with n=3-6 replicates per group. Data were analyzed using two-way ANOVA with Šidák’s multiple comparisons test. ns = not significant, **** = p<0.0001.

Conservation of the RHIM sequence determines RIPK3 NF-κB activation. A) Residues evolving under positive selection in primate RIPK3 and the primate RIPK3 RHIM domain mapped on the human sequence. (B) Residues evolving under positive selection in bat and carnivore RIPK3, mapped on to the Sturnira hondurensis and Felis catus sequences respectively. (C) Alignments of the RIPK3 RHIM across diverse vertebrates. Residue numbers refer to the human sequence. (D-E) Mammalian (D) and non-mammalian (E) RIPK3 proteins were transfected into WT HEK293T cells along with NF-κB firefly luciferase and control renilla luciferase reporter plasmids (see Materials and Methods). NF-κB activity was measured at 18h post-transfection. Species shown are mouse (Mus musculus), cat (Felis catus), pig (Sus scrofa), lizard (Anolis carolinensis), turtle (Chelonia mydas), and lamprey (Petromyzon marinus). Data are representative of 3-5 independent experiments with n=3-6 replicates per group. Data were analyzed using two-way ANOVA with Šidák’s multiple comparisons test. ns = not significant, **** = p<0.0001.

NF-κB activation is a shared function of RHIM-containing proteins and can be tuned by the RHIM. (A) Alignment of RIPK1 RHIM across diverse vertebrates. Residue numbers refer to the human sequence. (B) Diverse vertebrate RIPK1CT proteins. were transfected into HEK293T cells along with NF-κB firefly luciferase and control renilla luciferase reporter plasmids (see Materials and Methods), and NF-κB activity was measured at 18h post-transfection. (C) Alignment of ZBP1 RHIMs across diverse vertebrates. Residue numbers refer to the human sequence. (D) Activation of NF-κB by WT and RHIM mutant ZBP1 proteins. (E) NF-κB activation by human RIPK3 with the indicated RHIM tetrad variant. (F) Human RIPK3 proteins with the indicated RHIM tetrad variants were transfected into HEK293T cells with MLKL (gray circles) or MLKL and ZBP1 (orange squares) and viability was measured at 18h post-transfection. Data are representative of 2-5 independent experiments with n=3-6 replicates per group. Data were analyzed using two-way ANOVA with Šidák’s multiple comparisons test (A, D) one-way ANOVA with Tukey’s multiple comparisons test (E), or two-way ANOVA with Tukey’s multiple comparisons test (F). ns = not significant, **** = p<0.0001.