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An epistatic ratchet constrains the direction of glucocorticoid receptor evolution


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.

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Figure 1: Evolution and reversibility of glucocorticoid receptor function.
Figure 2: Identification of restrictive substitutions that impede reversibility.
Figure 3: Restrictive substitutions impede evolutionary reversibility.
Figure 4: Epistasis limits trajectories of reverse and forward evolution.

Accession codes

Primary accessions

Protein Data Bank

Data deposits

The atomic coordinates and structure factors for AncGR2 have been submitted to the Protein Data Bank (PDB) under accession number 3GN8.


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Supported by National Science Foundation IOB-0546906, National Institutes of Health R01-GM081592 and F32-GM074398, and a Sloan Foundation Fellowship to J.W.T. We thank M. Harms and members of the Thornton, Cresko and Phillips laboratories for comments.

Author Contributions J.T.B. and J.W.T. conceived the experiments. J.T.B. performed the functional experiments, E.A.O. the structural analysis, and J.W.T. the phylogenetic analysis. J.T.B., E.A.O. and J.W.T. interpreted the results. J.T.B. and J.T. wrote the paper.

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Correspondence to Joseph W. Thornton.

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Bridgham, J., Ortlund, E. & Thornton, J. An epistatic ratchet constrains the direction of glucocorticoid receptor evolution. Nature 461, 515–519 (2009).

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