The structural mechanisms by which proteins have evolved new functions are known only indirectly. We report x-ray crystal structures of a resurrected ancestral protein—the ∼450 million-year-old precursor of vertebrate glucocorticoid (GR) and mineralocorticoid (MR) receptors. Using structural, phylogenetic, and functional analysis, we identify the specific set of historical mutations that recapitulate the evolution of GR's hormone specificity from an MR-like ancestor. These substitutions repositioned crucial residues to create new receptor-ligand and intraprotein contacts. Strong epistatic interactions occur because one substitution changes the conformational position of another site. “Permissive” mutations—substitutions of no immediate consequence, which stabilize specific elements of the protein and allow it to tolerate subsequent function-switching changes—played a major role in determining GR's evolutionary trajectory.

Whether evolution can go back to an ancestral structure just by reversing the selection pressure on function has been a long-standing issue, but one hard to address based on just the history of forms. Bridgham et al. have now physically reconstituted ancient versions of a regulatory protein (the glucocorticoid receptor) and dissected the structural constraints imposed on the evolution of their function (which hormone they bind) at atomic resolution. They find that amino acids that were essential in an ancestral protein become neutral in a more recent form, where they are then subject to erosion by genetic drift. This loss deprives natural selection of the necessary raw material with which to reverse the historical substitutions — they are no longer 'adaptive' as they were in the other direction. Evolutionarily speaking, there is no turning back. Whether evolution can go back to an ancestral structure by reversing the selection pressure on function has long fascinated biologists. Here, the evolution of hormone specificity in the vertebrate glucocorticoid receptor is used as a case-study to investigate this issue; the mutations that optimized the new specificity of the glucocorticoid receptor are found to have destabilized elements of the protein structure that were required to support the ancestral conformation. The extent to which evolution is reversible has long fascinated biologists1,2,3,4,5,6,7,8. Most previous work on the reversibility of morphological and life-history evolution9,10,11,12,13 has been indecisive, because of uncertainty and bias in the methods used to infer ancestral states for such characters14,15. Further, despite theoretical work on the factors that could contribute to irreversibility1,8,16, 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 available3,8,17. 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.