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Fig. 6 | BMC Evolutionary Biology

Fig. 6

From: Resurrecting ancestral structural dynamics of an antiviral immune receptor: adaptive binding pocket reorganization repeatedly shifts RNA preference

Fig. 6

Two complex substitutions are sufficient to recapitulate the observed shift in RNA-binding preference following the earliest RLR gene duplication. We reconstructed ancestral RD protein sequences before and after the first RLR gene duplication (see Fig. 1), constructed structural models of RDs bound to blunt-ended and 5′ppp dsRNAs and performed replicate molecular dynamics simulations to characterize the structural basis for RNA binding (see Methods). We introduced the historical ΔEK47TEE and K88S substitutions into the ancRLR background as well as the ‘reverse’ TEE47ΔEK substitution into the ancMDA5/LGP2a background. We show the central structures of each RD-RNA interaction from replicate molecular dynamics simulations, with electrostatic potential (kT/e) displayed on the molecular surface. Residues forming hydrogen bonds to the RNA molecule in at least 50 % of sampled time points from molecular dynamics simulations (see Additional file 1: Figures S19 and S20) are shown as sticks, with dashed yellow lines indicating hydrogen bonds. We measured steady-state (Kd) and initial (Km) RNA-binding affinities of ancestral and mutant RDs bound to each RNA type (see Methods). We plot –log-transformed binding affinities, with longer bars indicating tighter affinity. Standard errors over three replicates are indicated

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