Crystal Structure of an Ancient Protein: Evolution by Conformational Epistasis Eric A. Ortlund, et al. Science 317, 1544 (2007); DOI: 10.1126/science.1142819

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Science (print ISSN 0036-8075; online ISSN 1095-9203) is published weekly, except the last week in December, by the American Association for the Advancement of Science, 1200 New York Avenue NW, Washington, DC 20005. Copyright 2007 by the American Association for the Advancement of Science; all rights reserved. The title Science is a registered trademark of AAAS.

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proaches have been and are being considered. For example, in Singapore, where 84% of the popu- lation lives in public housing (35), regulations that explicitly recognize the role of spatial segregation in sectarianism specify the percentage of ethnic groups to occupy housing blocks (36). This legally compels ethnic mixing at a scale finer than that which our study finds likely to lead to violence. Given the natural tendency toward social separa- tion, maintaining such mixing requires a level of authoritarianism that might not be entertained in other locations. Still, despite social tensions (37), the current absence of violence provides some support to our analysis. The alternative approach— aiding in the separation process by establishing clear boundaries between cultural groups to prevent violence—has also gained recent atten- tion (38, 39). Although further studies are needed, there exist assessments (39) of the impact of historical partitions in Ireland, Cyprus, the Indian subcontinent, and the Middle East that may be consistent with the understanding of type separation and a critical scale of mixing or separation presented here.

The insight provided by this study may help inform policy debates by guiding our understanding of the consequences of policy alternatives. The purpose of this paper does not include promoting specific policy options. Although our work re- inforces suggestions to consider separation, we are not diminishing the relevance of concerns about the desirability of separation or its process. Even where separation may be indicated as a way of preventing violence, caution is warranted to ensure that the goal of preventing violence does not become a justification for violence. Moreover, even a peaceful process of separation is likely to be objectionable. There may be ways to positively motivate separation using incentives, as well as to mitigate negative aspects of separation that often include displacement of populations and mobility barriers.

Our results for the range of filter diameters that provide good statistical agreement between reported and predicted violence in the former Yugoslavia and India suggest that regions of width less than 10 km or greater than 100 km may provide sufficient mixing or isolation to reduce the chance of violence. These bounds may be affected by a variety of secondary factors including social and economic conditions; the simulation resolu- tion may limit the accuracy of the lower limit; and boundaries such as rivers, other physical barriers, or political divisions will surely play a role. Still, this may provide initial guidance for strategic planning. Identifying the nature of boundaries to be established and the means for ensuring their stability, however, must reflect local issues.

Our approach does not consider the relative merits of cultures, individual acts, or immediate causes of violence, but rather the conditions that may promote violence. It is worth considering whether, in places where cultural differentiation is taking place, conflict might be prevented or minimized by political acts that create appropriate boundaries suited to the current geocultural regions rather than the existing

historically based state boundaries. Such bounda- ries need not inhibit trade and commerce and need not mark the boundaries of states, but should allow each cultural group to adopt independent behav- iors in separate domains. Peaceful coexistence need not require complete integration.

References and Notes 1. M. White, Deaths by Mass Unpleasantness: Estimated

Total for the Entire 20th Century, http://users.erols.com/ mwhite28/warstat8.htm (September 2005).

2. D. L. Horowitz, Ethnic Groups in Conflict (Univ. of California Press, Berkeley and Los Angeles, ed. 2, 2000).

3. B. Harff, T. R. Gurr, Ethnic Conflict in World Politics (Westview, Boulder, ed. 2, 2004).

4. S. Huntington, The Clash of Civilizations and the Remaking of World Order (Simon & Schuster, New York, 1996).

5. D. Chirot, M. E. P. Seligman, Eds., Ethnopolitical Warfare: Causes, Consequences, and Possible Solutions (American Psychological Association, Washington, DC, 2001).

6. M. Reynal-Querol, J. Conflict Resolut. 46, 29 (2002). 7. T. R. Gulden, Politics Life Sciences 21, 26 (2002). 8. H. Buhaug, S. Gates, J. Peace Res. 39, 417 (2002). 9. A. Varshney, Ethnic Conflict and Civic Life: Hindus and

Muslims in India (Yale Univ. Press, New Haven, CT, 2003). 10. M. D. Toft, The Geography of Ethnic Violence: Identity,

Interests, and the Indivisibility of Territory (Princeton Univ. Press, Princeton, NJ, 2003).

11. J. Fox, Religion, Civilization, and Civil War: 1945 through the New Millennium (Lexington Books, Lanham, MD, 2004).

12. M. Mann, The Dark Side of Democracy: Explaining Ethnic Cleansing (Cambridge Univ. Press, New York, 2004).

13. I. Lustick, Am. Polit. Sci. Rev. 98, 209 (2004). 14. Materials and methods are available as supporting

material on Science Online. 15. T. C. Schelling, J. Math. Sociol. 1, 143 (1971). 16. J. Mimkes, J. Therm. Anal. 43, 521 (1995). 17. H. P. Young, Individual Strategy and Social Structure

(Princeton Univ. Press, Princeton, NJ, 1998). 18. R. Van Kempen, A. S. Ozuekren, Urban Stud. 35, 1631 (1998). 19. Y. Bar-Yam, in Dynamics of Complex Systems (Perseus

Press, Cambridge, MA, 1997), chap. 7. 20. A. J. Bray, Adv. Phys. 43, 357 (1994). 21. I. M. Lifshitz, V. V. Slyozov, J. Phys. Chem. Solids 19, 35

(1961). 22. D. A. Huse, Phys. Rev. B 34, 7845 (1986).

23. W. Easterly, R. Levine, Q. J. Econ. 112, 1203 (1997). 24. P. Collier, A. Hoeffler, Oxf. Econ. Pap. 50, 563 (1998). 25. R. H. Bates, Am. Econ. Rev. 90, 131 (2000). 26. J. D. Fearon, D. D. Laitin, Am. Pol. Sci. Rev. 97, 75 (2003). 27. D. N. Posner, Am. J. Pol. Sci. 48, 849 (2004). 28. I. Daubechies, Ten Lectures on Wavelets, (SIAM,

Philadelphia, 1992). 29. A. Arneodo, E. Bacry, P. V. Graves, J. F. Muzy, Phys. Rev.

Lett. 74, 3293 (1995). 30. P. Ch. Ivanov et al., Nature 383, 323 (1996). 31. Map of Yugoslavia, Courtesy of the University of Texas

Libraries., www.lib.utexas.edu/maps/europe/yugoslav.jpg. 32. R. Petrovic, Yugosl. Surv. 33, 3 (1992). 33. K. Chaudhuri, Frontline 18 (no. 2), www.hinduonnet.com/

fline/fl1802/18020330.htm. 34. Final Report, Carnegie Commission on Preventing Deadly

Conflict, www.wilsoncenter.org/subsites/ccpdc/pubs/ rept97/finfr.htm.

35. A. Brief Background, Housing and Development Board, Singapore Government, www.hdb.gov.sg/fi10/fi10296p.nsf/ WPDis/About%20UsA%20Brief%20Background%20-% 20HDB's%20Beginnings.

36. Ethnic Integration Policy, Housing and Development Board, Singapore Government, www.hdb.gov.sg/fi10/ fi10201p.nsf/WPDis/Buying%20A%20Resale% 20FlatEthnic%20Group%20Eligibility.

37. D. Murphy, Christian Science Monitor, 5 February 2002, www.csmonitor.com/2002/0205/p07s01-woap.html.

38. J. Tullberg, B. S. Tullberg, Politics Life Sciences 16, 237 (1997). 39. C. Kaufmann, Int. Secur. 23, 120 (1998). 40. We thank G. Wolfe, M. Woolsey, and L. Burlingame for

editing the manuscript; B. Wang for assistance with figures; M. Nguyen and Z. Bar-Yam for assistance with identifying data; and I. Epstein, S. Pimm, F. Schwartz, E. Downs, and S. Frey for helpful comments. We acknowledge internal support by the New England Complex Systems Institute and the U.S. government for support of preliminary results.

Supporting Online Material www.sciencemag.org/cgi/content/full/317/5844/1540/DC1 Methods Figs. S1.1 to S4.3 SOM Text Table S1 References Bibliography

30 November 2006; accepted 13 August 2007 10.1126/science.1142734

Crystal Structure of an Ancient Protein: Evolution by Conformational Epistasis Eric A. Ortlund,1* Jamie T. Bridgham,2* Matthew R. Redinbo,1 Joseph W. Thornton2†

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.

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A central goal in molecular evolution is to understand the mechanisms and dynam- ics by which changes in gene sequence

generate shifts in function and therefore pheno- type (1, 2). A complete understanding of this

process requires analysis of how changes in protein structure mediate the effects of mutations on function. Comparative analyses of extant proteins have provided indirect insights into the diversifi- cation of protein structure (3–6), and protein

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engineering studies have elucidated structure- function relations that shape the evolutionary process (7–11). To directly identify the mecha- nisms by which historical mutations generated new functions, however, it is necessary to compare proteins through evolutionary time.

Here we report the empirical structures of an ancient protein, which we “resurrected” (12) by phylogenetically determining its maximum likeli- hood sequence from a large database of extant se- quences, biochemically synthesizing a gene coding for the inferred ancestral protein, expressing it in cultured cells, and determining the protein’s structure by x-ray crystallography. Specifically, we investigated the mechanistic basis for the functional evolution of the glucocorticoid receptor (GR), a hormone-regulated transcription factor present in all jawed vertebrates (13). GR and its sister gene, the mineralocorticoid receptor (MR), descend from the duplication of a single ancient gene, the ancestral corticoid receptor (AncCR), deep in the vertebrate lineage ~450 million years ago (Ma) (Fig. 1A) (13). GR is activated by the adrenal steroid cortisol and regulates stress response, glucose homeostasis, and other functions (14). MR is activated by aldosterone in tetrapods and by deoxycorticosterone (DOC) in teleosts to control electrolyte homeostasis, kidney

1Department of Chemistry, University of North Carolina, Chapel Hill, NC 27599, USA. 2Center for Ecology and Evolutionary Biology, University of Oregon, Eugene, OR 97403, USA.

*These authors contributed equally to this work. †To whom correspondence should be addressed. E-mail: [email protected]

Fig. 1. (A) Functional evolution

and colon function, and other processes (14). MR is also sensitive to cortisol, though considerably less so than to aldosterone and DOC (13, 15). Previously, AncCR was resurrected and found to have MR-like sensitivity to aldosterone, DOC, and cortisol, indicating that GR’s cortisol specificity is evolutionarily derived (13).

To identify the structural mechanisms by which GR evolved this new function, we used x-ray crystallography to determine the structures of the resurrected AncCR ligand-binding domain (LBD) in complex with aldosterone, DOC, and cortisol (16) at 1.9, 2.0, and 2.4 Å resolution, respectively (table S1). All structures adopt the classic active conformation for nuclear receptors (17), with unambiguous electron density for each hormone (Fig. 1B and figs. S1 and S2). AncCR’s structure is extremely similar to the human MR [root mean square deviation (RMSD) = 0.9 Å for all backbone atoms] and, to a lesser extent, to the human GR (RMSD = 1.2 Å). The network of hydrogen-bonds supporting activation in the human MR (18) is present in AncCR, indicating that MR’s structural mode of action has been conserved for >400 million years (fig. S3).

Because aldosterone evolved only in the tetrapods, tens of millions of years after AncCR, that receptor’s sensitivity to aldosterone was surprising (13). The AncCR-ligand structures indicate that the receptor’s ancient response to aldosterone was a structural by-product of its sensitivity to DOC, the likely ancestral ligand, which it binds almost identically (Fig. 1C). Key contacts for binding DOC involve conserved

surfaces among the hormones, and no obligate contacts are made with moieties at C11, C17, and C18, the only variable positions among the three hormones. These inferences are robust to uncer- tainty in the sequence reconstruction: We modeled each plausible alternate reconstruction [posterior probability (PP) > 0.20] into the AncCR crystal structures and found that none significantly af- fected the backbone conformation or ligand inter- actions. The receptor, therefore, had the structural potential to be fortuitously activated by aldoster- one when that hormone evolved tens of millions of years later, providing the mechanism for evo- lution of the MR-aldosterone partnership by mo- lecular exploitation, as described (13).

To determine how GR’s preference for cortisol evolved, we identified substitutions that occurred during the same period as the shift in GR function. We used maximum likelihood phylogenetics to de- termine the sequences of ancestral receptors along the GR lineage (16). The reconstructions had strong support, with mean PP >0.93 and the vast majority of sites with PP >0.90 (tables S2 and S3). We synthesized a cDNA for each reconstructed LBD, expressed it in cultured cells, and experimentally characterized its hormone sensitivity in a reporter gene transcription assay (16). GR from the com- mon ancestor of all jawed vertebrates (AncGR1 in Fig. 1A) retained AncCR’s sensitivity to aldoster- one, DOC, and cortisol. At the next node, however, GR from the common ancestor of bony vertebrates (AncGR2) had a phenotype like that of modern GRs, responding only to cortisol. This inference is robust to reconstruction uncertainty: We introduced

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of corticosteroid receptors. Dose- response curves show transcrip- tion of a luciferase reporter gene by extant and resurrected ances- tral receptors with varying doses (in log M) of aldosterone (green), DOC (orange), and cortisol (pur- ple). Black box indicates evolution of cortisol specificity. The number of sequence changes on each branch is shown (aa, replacement; D, deletion). Scale bars, SEM of three replicates. Node dates from the fossil record (19, 20). For com- plete phylogeny and sequences, see fig. S10 and table S5. (B) Crystal structure of the AncCR LBD with bound aldosterone (green, with red oxygens). Helices are la- beled. (C) AncCR’s ligand-binding pocket. Side chains (<4.2 Å from bound ligand) are superimposed from crystal structures of AncCR with aldosterone (green), DOC (orange), and cortisol (purple). Oxygen and nitrogen atoms are red and blue, respectively; dashed lines indicate hydrogen bonds. Arrows show C11, C17, and C18 positions, which differ among the h

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plausible alternative states by mutagenesis, but none changed function (fig. S4). GR’s specificity therefore evolved during the interval between these two speciation events, ~420 to 440 Ma (19, 20).

During this interval, there were 36 substitutions and one single-codon deletion (figs. S5 and S6). Four substitutions and the deletion are conserved in one state in all GRs that descend from AncGR2 and in another state in all receptors with the ancestral function. Two of these—S106P and L111Q (21)— were previously identified as increasing cortisol specificity when introduced into AncCR (13). We introduced these substitutions into AncGR1 and found that they recapitulate a large portion of the functional shift from AncGR1 to AncGR2, radi- cally reducing aldosterone and DOC response while maintaining moderate sensitivity to cortisol (Fig. 2A); the concentrations required for half- maximal activation (EC50) by aldosterone and DOC increased by 169- and 57-fold, respectively, whereas that for cortisol increased only twofold. A strong epistatic interaction between substitutions was apparent: L111Q alone had little effect on sensitivity to any hormone, but S106P dramatically reduced activation by all ligands. Only the combination switched receptor preference from aldosterone and DOC to cortisol. Introducing these historical substitutions into the human MR yielded a completely nonfunctional receptor, as did reversing them in the human GR (fig. S7). These results emphasize the importance of having the ancestral sequence to reveal the functional impacts of historical substitutions.

To determine the mechanism by which these two substitutions shift function, we compared the structures of AncGR1 and AncGR2, which were generated by homology modeling and energy minimization based on the AncCR and human GR crystal structures, respectively (16). These structures are robust to uncertainty in the recon- struction: Modeling plausible alternate states did not significantly alter backbone conformation, interactions with ligand, or intraprotein interactions. The major structural difference between AncGR1

Fig. 2. Mechanism for switching A AncGR1’s ligand preference from al-

and AncGR2 involves helix 7 and the loop preceding it, which contain S106P and L111Q and form part of the ligand pocket (Fig. 2B and fig. S8). In AncGR1 and AncCR, the loop’s position is stabilized by a hydrogen bond between Ser106 and the backbone carbonyl of Met103 . Replacing Ser106

with proline in the derived GRs breaks this bond and introduces a sharp kink into the backbone, which pulls the loop downward, repositioning and partially unwinding helix 7. By destabilizing this crucial region of the receptor, S106P impairs activation by all ligands. The movement of helix 7, however, also dramatically repositions site 111, bringing it close to the ligand. In this conforma- tional background, L111Q generates a hydrogen bond with cortisol’s C17-hydroxyl, stabilizing the receptor-hormone complex. Aldosterone and DOC lack this hydroxyl, so the new bond is cortisol- specific. The net effect of these two substitu- tions is to destabilize the receptor complex with aldosterone or DOC and restore stability in a cortisol-specific fashion, switching AncGR2’s pref- erence to that hormone. We call this mode of structural evolution conformational epistasis, be- cause one substitution remodels the protein back- bone and repositions a second site, changing the functional effect of substitution at the latter.

Although S106P and L111Q (“group X” for convenience) recapitulate the evolutionary switch in preference from aldosterone to cortisol, the receptor retains some sensitivity to MR’s ligands, unlike AncGR2 and extant GRs. We hypothesized that the other three strictly conserved changes that occurred between AncGR1 and AncGR2 (L29M, F98I, and deletion S212D) would complete the functional switch. Surprisingly, introducing these “group Y” changes into the AncGR1 and AncGR1 + X backgrounds produced completely nonfunc- tional receptors that cannot activate transcription, even in the presence of high ligand concentrations (Fig. 3A). Additional epistatic substitutions must have modulated the effect of group Y, which pro- vided a permissive background for their evolution that was not yet present in AncGR1.

The AncCR crystal structure allowed us to identify these permissive mutations by analyzing the effects of group Y substitutions (Fig. 3B). In all steroid receptors, transcriptional activity depends on the stability of an activation-function helix (AF-H), which is repositioned when the ligand binds, generating the interface for tran- scriptional coactivators. The stability of this orientation is determined by a network of inter- actions among three structural elements: the loop preceding AF-H, the ligand, and helix 3 (17). Group Y substitutions compromise activation be- cause they disrupt this network. S212D eliminates a hydrogen bond that directly stabilizes the AF-H loop, and L29M on helix 3 creates a steric clash and unfavorable interactions with the D-ring of the hormone. F98I opens up space between helix 3, helix 7, and the ligand; the resulting instability is transmitted indirectly to AF-H, impairing activation by all ligands (Fig. 3B). If the protein could tolerate group Y, however, the structures predict that these mutations would enhance cortisol specificity: L29M forms a hydrogen bond with cortisol’s unique C17-hydroxyl, and the additional space created by F98I relieves a steric clash between the repositioned loop and Met108 , stabilizing the key interaction between Q111 and the C17-hydroxyl (Fig. 3B).

We hypothesized that historical substitutions that added stability to the regions destabilized by group Y might have permitted the evolving pro- tein to tolerate group Y mutations and to complete the GR phenotype. Structural analysis suggested two candidates (group Z): N26T generates a new hydrogen bond between helix 3 and the AF-H loop, and Q105L allows helix 7 to pack more tightly against helix 3, stabilizing the latter and, indirectly, AF-H (Fig. 3B). As predicted, intro- ducing group Z into the nonfunctional AncGR1 + X + Y receptor restored transcriptional activity, indicating that Z is permissive for Y (Fig. 3A). Further, AncGR1 + X + Y + Z displays a fully GR-like phenotype that is unresponsive to aldosterone and DOC and maintains moderate

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dosterone to cortisol. (A) Effect of substitutions S106P and L111Q on the resurrected AncGR1’s response to hor- mones. Dashed lines indicate sensitivity

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to aldosterone (green), cortisol (purple), and DOC (orange) as the EC50 for reporter gene activation. Green arrow shows probable pathway through a functional intermediate; red arrow, intermediate with radically reduced sensitivity to all hormones. (B) Struc- tural change conferring new ligand specificity. Backbones of helices 6 and 7 from AncGR1 (green) and AncGR2 (yellow) in complex with cortisol are superimposed. Substitution S106P Hormone (log M) induces a kink in the interhelical loop of AncGR2, repositioning sites 106 and 111 (arrows). In this background, L111Q forms a new hydrogen bond with cortisol’s unique C17-hydroxyl (dotted red line).

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cortisol sensitivity. Both N26T and Q105L are required for this effect (table S4). Strong epistasis is again apparent: Adding group Z substitutions in the absence of Y has little or no effect on ligand- activated transcription, presumably because the receptor has not yet been destabilized (Fig. 3A). Evolutionary trajectories that pass through func- tional intermediates are more likely than those involving nonfunctional steps (22), so the only historically likely pathways to AncGR2 are those in which the permissive substitutions of group Z and the large-effect mutations of group X occurred before group Y was complete (Fig. 3C).

Fig. 3. Permissive substitutions in the evolution of receptor specificity. (A) Effects of various combinations of historical substitutions on AncGR1’s transcriptional activity and hormone- sensitivity in a reporter gene assay. Group Y (L29M, F98I, and S212D) abol- ishes receptor activity unless groups X (S106P, L111Q) and Z (N26T and Q105L) are present; the XYZ combina- tion yields complete cortisol-specificity. The 95% confidence interval for each EC50 is in parentheses. Dash, no acti- vation. (B) Structural prediction of permissive substitutions. Models of

AncGR1 (green) and AncGR2 (yellow) are shown with cortisol. Group X and Y substitutions (circles and rectangles) yield new interactions with the C17- hydroxyl of cort