Fig. 4. Structural identification of an ancient permissive substitution. (A) Comparison of the structures of AncCR (blue) and AncGR2 (yellow). Y27R generates a novel cation-p interaction in AncGR2 (dotted cyan line), replacing the weaker ancestral hydrogen bond (dotted red) and imparting additional stability to helix 3. (B) Y27R is permissive for the substitutions that confer GR function. Reporter gene activation by AncGR1 + XYZ (upper right) is abolished when Y27R is reversed (lower right). (Left) Y27R has negligible effect in the AncCR background (or in AncGR1, fig. S9). Green, orange, and purple lines show aldosterone, DOC, and cortisol responses, respectively. Green arrows, likely pathway through functional intermediates.