Fig. 2.
Functional effects of historical substitutions are robust to incorporation of uncertainty. (A) The s36P substitution (upper case, derived; lower case, ancestral) changes the function of Anc-gkdup from a nucleotide kinase (orange squares, plotted using the y-axis at left) to a protein binding domain (grey diamonds, plotted using the right y-axis), in both the ML and AltAll backgrounds. Error bars indicate SEM from three independent experiments. Colored lines show the effect of the mutation on each function. (B) Function switching mutations (+F) switch the response element specificity of AncSR1DBD from ERE (green squares) to SRE (purple circles) in both the ML or AltAll ancestral backgrounds. (C) Introducing two major-effect historical substitutions into either AncSR1LBD ML or AltAll reconstructions decreases preference for an aromatized steroid (magenta with Lewis structure shown), and increases sensitivity to a non-aromatized steroid (blue). Points and error bars shown mean and SEM of EC50s from three independent dose-response experiments. (D) Reversal of the two key historical substitutions to their ancestral states (upper case, derived; lower case, ancestral) switches the preference of the ML and AltAll AncSR2LBDs from non-aromatized (blue) to aromatized (pink) steroids. Some data in this figure were previously reported in Anderson et al. 2016, McKeown et al. 2014, Eick et al. 2012, and Harms et al. 2013.