Evolution of minimal specificity and promiscuity in steroid hormone receptors

Abstract

Most proteins are regulated by physical interactions with other molecules; some are highly specific, but others interact with many partners. Despite much speculation, we know little about how and why specificity/promiscuity evolves in natural proteins. It is widely assumed that specific proteins evolved from more promiscuous ancient forms and that most proteins' specificity has been tuned to an optimal state by selection. Here we use ancestral protein reconstruction to trace the evolutionary history of ligand recognition in the steroid hormone receptors (SRs), a family of hormone-regulated animal transcription factors. We resurrected the deepest ancestral proteins in the SR family and characterized the structure-activity relationships by which they distinguished among ligands. We found that that the most ancient split in SR evolution involved a discrete switch from an ancient receptor for aromatized estrogens--including xenobiotics--to a derived receptor that recognized non-aromatized progestagens and corticosteroids. The family's history, viewed in relation to the evolution of their ligands, suggests that SRs evolved according to a principle of minimal specificity: at each point in time, receptors evolved ligand recognition criteria that were just specific enough to parse the set of endogenous substances to which they were exposed. By studying the atomic structures of resurrected SR proteins, we found that their promiscuity evolved because the ancestral binding cavity was larger than the primary ligand and contained excess hydrogen bonding capacity, allowing adventitious recognition of larger molecules with additional functional groups. Our findings provide an historical explanation for the sensitivity of modern SRs to natural and synthetic ligands--including endocrine-disrupting drugs and pollutants--and show that knowledge of history can contribute to ligand prediction. They suggest that SR promiscuity may reflect the limited power of selection within real biological systems to discriminate between perfect and "good enough."

Figure 1. Evolutionary expansion of the steroid receptors and their ligands.

A, Pathway for synthesis of vertebrate steroid hormones. The main pathway – synthesis of estrogens (red) via progestagens (blue) and androgens (green) – is at least as ancient as the chordate ancestor. Yellow box, synthesis pathway to corticosteroids (purple), is a later evolutionary novelty found only in vertebrates. The numbering system on the steroid backbone is shown in black. B, Phylogeny of the SR gene family. Receptors are color-coded by the classes of ligands to which they are most sensitive. Ancestral steroid receptors (AncSR1 and AncSR2) resurrected in this study are marked as circles. The number of sequences in each clade is shown in parentheses. Branch supports show approximate likelihood ratios and chi-square confidence metrics for each clade compared to the best phylogeny without that clade. Estrogen-responsive receptors are shown in red. For unreduced phylogenies and a list of sequences, see Figures S10, S11 and Table S7. C, Maximum likelihood reconstruction of ligand-contacting amino acids in AncSR1 and AncSR2, along with residues at homologous sites in extant human SRs. The steroid rings are labeled; circled R indicates polar functional groups at which the major steroid classes differ from each other; arrows indicate residues within hydrogen bonding distance. Residues that differ between AncSR1 and AncSR2 are highlighted in yellow.

Figure 2. Ligand-recognition rules of AncSR1 and AncSR2.

A, The sensitivity of AncSR1-LBD (top panel) and AncSR2-LBD (bottom panel) to various hormones (Table S4) was characterized in a triplicate luciferase reporter assay and is displayed as EC50, the concentration at which half-maximal reporter activation is achieved. Error bars, 95% confidence interval. Sets of hormones are grouped by color and are numerically labeled according to the list below. B, AncSR1's ligand recognition criteria. Each pair of bars shows the EC50 of AncSR1 to a pair of hormones that differ only by aromatization of the A-ring (shown in red on the ligand structure and in the key). Unlike aromatization, substitution of a 17-keto or acetyl for estradiol's hydroxyl has only a weak effect on sensitivity, as shown by the small differences among pairs. C-I, AncSR2's ligand recognition criteria. Each pair of bars shows the sensitivity of the receptor to hormones that differ only in the functional group at specified positions or aromatization of the A-ring. Bar labels indicate the substance tested: 0, cholesterol, 1, 11-deoxycorticosterone, 2, 11-deoxycortisol; 3, corticosterone; 4, cortisol; 5, aldosterone; 6, progesterone; 7, 17α-hydroxyprogesterone; 8, 19-norprogesterone; 9, 4-pregnenolone; 10, 5-pregnenolone; 11, 20α hydroxyprogesterone; 12, 20β hydroxyprogesterone; 13, testosterone; 14, dihydrotestosterone; 15, 4-androstenediol; 16, 5-androstenediol; 17, 19-nortestosterone; 18, bolandiol; 19, estradiol; 20, estrone; 21, estriol; 22, 4-androstenedione; 23, 19-nor-1, 3, 5(10)-pregnatriene-3-ol-20-one (NPT).

Figure 3. Evolution of minimal specificity.

A, Evolution of ligand-recognition criteria on the SR phylogeny. For each ancient and extant receptor, the criteria that distinguish activating ligands from other endogenous steroids are shown in brackets. Rules labeled “not” indicate significantly strongly reduced sensitivity when the specified moiety is present; other rules indicate strongly increased sensitivity when the moiety is present. The structures of representative endogenous hormones – estrogens (E), androgens (A), progestagens (P) and corticosteroids (C) – that were synthesized at each point in time are shown. Green portions of each hormone show moieties that satisfy the receptor's rules; red portions violate rules. Each receptor's rules are sufficient to allow activation by only a single class of hormones (gray boxes). The evolution of corticosteroid synthesis is indicated; AncSR2's criteria would not have been sufficient to distinguish corticosteroids from progestagens. Inset: common steroid structure with A-ring and key carbons labeled. Dose-response curves for extant receptors are shown in Figure S7. B, AncSR1 is activated/antagonized by xenoestrogens in a luciferase reporter assay. IC50, concentration at which half-maximal inhibition was achieved in the presence of estradiol (EC₈₀ = 200 nM). Each point shows the mean and SEM of three replicates.

Figure 4. Structural causes of minimal specificity.

A, X-ray crystal structures of AncSR2 with progesterone (blue) and DOC (purple) are superimposed. Ligands are shown as sticks. Helices making major ligand contacts and the activation-function helix (AF-H) are shown in contrasting colors. B, Structural causes of promiscuity in AncSR2. The ligand cavity of the AncSR2-progesterone structure, shown as a surface, has adequate volume to accommodate the 21-hydroxyl of DOC. Ligand contacts in the crystal structures of AncSR2 with progesterone (blue) and DOC (purple) are shown. Thick sticks, ligand; thin sticks, side chains that contact ligand; balls, α-carbons. Steroid carbons 11, 17, 20, and 21 are numbered. Hydrogen bonds are shown as orange dotted lines. C, Structural basis for promiscuity in AncSR1. Ligand contacts in the AncSR1 model with estradiol (magenta) and NPT (blue) are shown. The cavity of the AncSR1-estradiol complex, which has adequate room to accommodate the 17-acetyl of NPT, is shown. Two side chains between the viewer and the ligand are hidden for clarity.