An epistatic ratchet constrains the direction of glucocorticoid receptor evolution

Abstract

The extent to which evolution is reversible has long fascinated biologists. Most previous work on the reversibility of morphological and life-history evolution has been indecisive, because of uncertainty and bias in the methods used to infer ancestral states for such characters. Further, despite theoretical work on the factors that could contribute to irreversibility, there is little empirical evidence on its causes, because sufficient understanding of the mechanistic basis for the evolution of new or ancestral phenotypes is seldom available. By studying the reversibility of evolutionary changes in protein structure and function, these limitations can be overcome. Here we show, using the evolution of hormone specificity in the vertebrate glucocorticoid receptor as a case-study, that the evolutionary path by which this protein acquired its new function soon became inaccessible to reverse exploration. Using ancestral gene reconstruction, protein engineering and X-ray crystallography, we demonstrate that five subsequent 'restrictive' mutations, which optimized the new specificity of the glucocorticoid receptor, also destabilized elements of the protein structure that were required to support the ancestral conformation. Unless these ratchet-like epistatic substitutions are restored to their ancestral states, reversing the key function-switching mutations yields a non-functional protein. Reversing the restrictive substitutions first, however, does nothing to enhance the ancestral function. Our findings indicate that even if selection for the ancestral function were imposed, direct reversal would be extremely unlikely, suggesting an important role for historical contingency in protein evolution.

Fig. 1

Evolution and reversibility of GR function. a) Reduced phylogeny of corticosteroid receptors. Blue, receptors sensitive to both cortisol and mineralocorticoids; purple, sensitive to cortisol only; black, other steroid receptors (AR, androgen receptor; PR, progestagen receptor). Ancestral proteins AncGR1 and AncGR2 are labeled. 37 amino acid changes, including groups X, Y, and Z, occurred during the interval between these two proteins (black box; for complete list and alignment, see Fig. S1). Parentheses show number of sequences in each group. b, c) Ligand sensitivities of AncGR1 and AncGR2, shown as fold increase in expression of a luciferase reporter in the presence of increasing doses of cortisol (purple), aldosterone (solid blue), and deoxycorticosterone (DOC, dashed blue). Error bars, SEM. d) Conformational change causing cortisol-specificity in AncGR2 (see ref. 24). Partial structures of AncGR1 and AncGR2 are superimposed. Substitutions in group X (S106P and L111Q) are large effect mutations that reposition helix 7 (H7) and form a hydrogen bond to the 17-OH that is unique to cortisol (purple). Black arrows indicate change in position of these residues. Substitutions in groups Y (L29M, F98I, S212Δ), and Z (N26T, Q105L) optimize the derived function. e) When substitutions in sets X, Y, and Z are introduced into AncGR1, they recapitulate the evolution of a cortisol-specific activator. f) When these substitutions are reversed to the ancestral state (xyz) in the AncGR2 background, activation by all ligands is lost. g) All AncGR2 combinations in which group X is reversed also yield non-functional receptors.

Fig. 2

Identification of “restrictive” substitutions that impede reversibility. a) Group W residues are conserved in the AncGR1-like state in virtually all extant receptors that retain the ancestral function. b) X-ray crystal structure of AncGR2 (bronze) with dexamethasone (purple). Repositioned Helix 7 is shown in grey. Residues substituted between AncGR1 and AncGR2 are marked with spheres at the α-carbon. Cyan, candidate restrictive substitutions (group W). Sites in groups X, Y, and Z are shown in medium, dark, and light green, respectively. Blue, V234F. c) Ligand pockets of AncGR1 (green, with cortisol) and AncGR2 (bronze, with dexamethasone). Group w residues (cyan) in their ancestral state in AncGR1 are predicted to support the ancestral conformation of helix 7 (grey), but to destabilize that conformation in the derived states of AncGR2.

Fig. 3

Restrictive substitutions impede evolutionary reversibility. a) When group W substitutions are restored to their ancestral state (w), the nonfunctional AncGR2-xyz is rescued, and the ancestral sensitivity to all three ligands is restored. Fold increase in luciferase reporter expression is shown with cortisol (purple), aldosterone (solid blue), and DOC (dashed blue). b) Group W substitutions all impede reversiblility: restoring the ancestral states singly (Y107A) or in structurally interacting pairs (Q84H/C91Y and Q114G/M197L) partially rescues AncGR2-xyz.

Fig. 4

Epistasis limits trajectories of reverse and forward evolution. The corners of each hypercube represent states for residue sets X, Y, Z, and W. Edges show pathways between the derived (XYZW) and ancestral (xyzw) states. Red edges show unlikely evolutionary paths through nonfunctional intermediates; black paths pass through functional intermediates. Filled shapes at vertices indicate sensitivity to aldosterone (blue squares), DOC (blue circles), and cortisol (purple triangles); empty shapes, no activation by these hormones. Tables below each cube show sensitivity to each hormone as the EC50 (concentration required for half-maximal activation), with 95% confidence interval. Dashes, no activation. Asterisks, state combinations in AncGR2 and AncGR1. a) Limited evolutionary pathways to reverse AncGR2 (bronze) to the ancestral structure and function. Mutations were introduced in the AncGR2 background. b) Functional effect of substitutions during “forward” evolution when introduced into AncGR1 (green). The sets X, Y, Z, and W contain each more than one site, so the complete sequence space for each cube has 12 dimensions, 4096 vertices, and numerous additional trajectories.