Fig 7. Functional differentiation of ancestral RIG-I CARD1 and CARD2 domains occurred after the proliferation of RLR-IPS1 CARDs in deuterostomes, due to coevolution between RIG-I and IPS1 CARDs.

Ancestral RLR and IPS1 CARD domains were resurrected, and we measured the kinetics of RIG-I and MDA5 CARDs bound to IPS1 CARD before (A) and after (B) the RLR-IPS1 CARD proliferation in early deuterostomes (dotted vertical line on simplified CARD phylogeny) (Figs 3 and 5). We plot the mean and standard error of–log-transformed steady-state dissociation (pKd) and initial binding rate (pKm) when ancestral RLR CARD domains bind to IPS1 CARD before (A) and after (B) the deuterostome proliferation (see Materials and Methods). The approximate level of nonspecific binding is indicated by a vertical dotted line (inferred using human CASP9), and longer bars indicate tighter binding (S17 and S18 Figs).