Fig. 7

A single amino acid substitution is sufficient to recapitulate the observed re-evolution of high-affinity 5′ppp dsRNA binding between ancMDA5/LGP2a and ancMDA5/LGP2b. We reconstructed RD protein sequences of the first (ancMDA5/LGP2a) and last (ancMDA5/LGP2b) ancestral RLRs between the first and second major RLR gene duplications (see Fig. 1). We additionally introduced a single historical H63S substitution into the ancMDA5/LGP2a background. We show the central structures from replicate molecular dynamics simulations of each RD bound to blunt-ended and 5′ppp dsRNA (see Methods). Electrostatic potential (kT/e) is displayed across the molecular surface of each RNA-binding pocket (left panels). Residues forming hydrogen bonds to the RNA in at least 50 % of molecular dynamics time points (see Additional file 1: Figures S24 and S25) are shown as sticks, with dashed yellow lines indicating hydrogen bonds. We plot –log-transformed steady-state (Kd) and initial (Km) RD-RNA binding rates, with bars indicating standard errors. Right panels show each RD bound to 5′ppp dsRNA. A dotted line connects the H/S63 (green) and R102 (blue) residues and the zinc-binding pocket (teal)