N the A-ring conformation of the ligand. In the human PR the DHP A-ring thereby adopts a different position in the JI 101 web binding pocket affecting A-ring specific interactions. In the (��)-Hexaconazole site elephant PR, the different positioning of the DHP A-ring is blocked by the presence of the Ala722 methyl group, which would clash with the C1 of DHP and thereby pushes the DHP A-ring into a similar position than progesterone, explaining a similar binding affinity for both ligands. Apart from the change in specificity, we observed that the elephant PR has a 2.3-fold higher binding affinity towards progesterone and DHP compared to the human hPR 722A mutant. The higher affinity of the elephant compared to human PR was partly mediated by the S796P exchange. However we found that introducing the S796P substitution in the G722A mutated human receptor did not lead to a further increase in affinity for neither DHP, nor progesterone. This could be explained by the finding that Leu796 neighboring the S796P exchange and binding the D-ring should be most sensitive to changes in position 722. Enhanced rigidity by either G722A or S796P leads to enhanced affinity, while the second substitution has no further effect. Hence, a possible evolutionary scenario would be that the S796P mutation occurred first in order to balance the restricted availability of progesterone, while the G722A exchange and with it the possibility to efficiently use DHP appeared in a later step during evolution. This theory is further strengthened byElephant Progestin ReceptorElephant Progestin ReceptorFigure 7. The G722A exchange evolved 5 times during mammalian evolution. (A) Sequence alignment of residue 722 and surrounding amino acids of the PR LBD. (B) Phylogenetic tree of mammalian evolution deduced from Murphy et al. (16) and Killian et al. (17). Blue arrows indicate the substitution of G722 to alanine, green arrows the substitution to cysteine. (C) PR LBD DNA sequences from mammals listed in (B) were aligned and a codon analysis for positive/purifying selection performed based on phylogenetic relationships depicted in (B). Residues of elephant PR are color-coded according to their selective pressure during mammalian evolution. doi:10.1371/journal.pone.0050350.gthe fact, that also megabats acquired the 24195657 S796P exchange independently of Ala722. Both G722A and S796P substitutions also seem to be responsible for the different affinity profile of MGA, which is more bulky than progesterone. While the longer side chains of MGA result in a higher affinity to the human receptor, in the elephant PR they cause sterical clashes and thus drastically reduce affinity. Steroid hormone receptors evolved under the principle of molecular exploitation [32]. The PR developed as a result of two rounds of gene duplication events, which starting from an ancestral estrogen receptor generated a functional progesterone and a corticosteroid receptor. As progesterone is an intermediate in the synthesis of estradiol, the duplicated receptor achieved specificity for a preexisting compound, known as “ligand first” model [32]. A third duplication event separated MR and GR from the ancestral corticosteroid receptor. In this case the ancestral receptor already had affinity for both mineralocorticoids and glucocorticoids, which was used by cortisol to build up a new receptor-hormone system, known as “receptor first” model [11]. For the evolution of ligand specificity of the elephant PR both “ligand first” and “receptor first” sc.N the A-ring conformation of the ligand. In the human PR the DHP A-ring thereby adopts a different position in the binding pocket affecting A-ring specific interactions. In the elephant PR, the different positioning of the DHP A-ring is blocked by the presence of the Ala722 methyl group, which would clash with the C1 of DHP and thereby pushes the DHP A-ring into a similar position than progesterone, explaining a similar binding affinity for both ligands. Apart from the change in specificity, we observed that the elephant PR has a 2.3-fold higher binding affinity towards progesterone and DHP compared to the human hPR 722A mutant. The higher affinity of the elephant compared to human PR was partly mediated by the S796P exchange. However we found that introducing the S796P substitution in the G722A mutated human receptor did not lead to a further increase in affinity for neither DHP, nor progesterone. This could be explained by the finding that Leu796 neighboring the S796P exchange and binding the D-ring should be most sensitive to changes in position 722. Enhanced rigidity by either G722A or S796P leads to enhanced affinity, while the second substitution has no further effect. Hence, a possible evolutionary scenario would be that the S796P mutation occurred first in order to balance the restricted availability of progesterone, while the G722A exchange and with it the possibility to efficiently use DHP appeared in a later step during evolution. This theory is further strengthened byElephant Progestin ReceptorElephant Progestin ReceptorFigure 7. The G722A exchange evolved 5 times during mammalian evolution. (A) Sequence alignment of residue 722 and surrounding amino acids of the PR LBD. (B) Phylogenetic tree of mammalian evolution deduced from Murphy et al. (16) and Killian et al. (17). Blue arrows indicate the substitution of G722 to alanine, green arrows the substitution to cysteine. (C) PR LBD DNA sequences from mammals listed in (B) were aligned and a codon analysis for positive/purifying selection performed based on phylogenetic relationships depicted in (B). Residues of elephant PR are color-coded according to their selective pressure during mammalian evolution. doi:10.1371/journal.pone.0050350.gthe fact, that also megabats acquired the 24195657 S796P exchange independently of Ala722. Both G722A and S796P substitutions also seem to be responsible for the different affinity profile of MGA, which is more bulky than progesterone. While the longer side chains of MGA result in a higher affinity to the human receptor, in the elephant PR they cause sterical clashes and thus drastically reduce affinity. Steroid hormone receptors evolved under the principle of molecular exploitation [32]. The PR developed as a result of two rounds of gene duplication events, which starting from an ancestral estrogen receptor generated a functional progesterone and a corticosteroid receptor. As progesterone is an intermediate in the synthesis of estradiol, the duplicated receptor achieved specificity for a preexisting compound, known as “ligand first” model [32]. A third duplication event separated MR and GR from the ancestral corticosteroid receptor. In this case the ancestral receptor already had affinity for both mineralocorticoids and glucocorticoids, which was used by cortisol to build up a new receptor-hormone system, known as “receptor first” model [11]. For the evolution of ligand specificity of the elephant PR both “ligand first” and “receptor first” sc.
