F Tyr777 and Phe778 respectively, for the hPR-P4 complex the resulting sum was 101 (Figure 4). However, in hPRDHP, the summarized hydrogen-bond populations were only 85 . In the elePR complexes with both P4 and DHP these values were much more similar with 90 and 95 , respectively. From our data we can conclude that the altered A-ring position in the hPR-DHP complex inhibitor affected the hydrogen bond network of the binding pocket, both reducing the probability of H-binding via Arg766 as well as destabilizing the intra-residual H-bonds of the binding pocket.Human and Elephant PR LBD Differ in 25033180 DHP PositioningTo identify structural differences in the binding of progesterone and DHP to human and elephant PR, we performed unrestrained molecular dynamics simulations based on all four receptor-ligand combinations. This allowed us to study ligand positioning as well as local receptor flexibilities. The closest-to-average structures of the trajectories in comparison to the X-ray structure are shown in Figure 3. While progesterone docked in a position similar to the Xray structure in both receptors (Figure 3A), DHP exhibited a slightly different orientation compared to the ligand in the crystalstructure (Figure 3B). The binding pocket is relatively restricted around the A-ring, leading to similar positions of the A-rings of both ligands. The ring geometries, however, differ between progesterone and DHP because of the double versus single bond between C4 and C5 (Figure 1). This causes a twist in the ligand leading to different positions of the remaining three rings of progesterone and DHP (seen best Figure 3C, center panels). Yet, the acyl group attached to the D-ring is found in a similar position in all four investigated complexes. Due to the altered geometry of the A-ring, there is no contact between C19 and the receptor. Therefore, DHP bound to hPR adopts a position with the A-ring that is closer to the H3 helix resulting in an A-ring orientation that is different from progesterone (Figure 3B). In the elePR-DHP complex this orientation is not possible, as the methyl side chain of Ala722 would clash with C1 of the ligand. As a result, the DHP Aring in the complex with elePR is shifted away from the H3 helix, adopting an orientation that is similar to that of the progesterone A-ring.Additional Amino Acid Substitutions Increase Ligand Affinities of the PR Accompanied by a Loss of Flexibility of 1326631 the Binding PocketWe next sought to analyze the molecular basis of the overall gain of ligand affinity that we have observed for elePR compared 
to hPR, by focusing on the additional amino acid differences between both species. Three amino acid exchanges were found in close vicinity to the residues involved in steroid binding: V698M,Elephant Progestin ReceptorElephant Progestin ReceptorFigure 3. In elePR alanine 722 orientates the DHP A-ring into a position similar to progesterone. (A,B) Structures of progesterone (P4) and DHP bound to the human and elephant PR LBD compared to the X-ray structure (PDB code 1a28) (green) generated by molecular-dynamics simulations. The frames of each Epigenetic Reader Domain trajectory were fitted on the start frame using the Ca atoms of the helical parts and resulting averaged coordinates were used for a further fit. Represented are the frames with the least relative deviation to the averaged coordinates. The contour lines represent the solvent/ligand-accessible parts of residue 722. (A) hPR-P4 (orange), elePR-P4 (violet). (B) hPR-DHP (yellow), elePR-DHP.F Tyr777 and Phe778 respectively, for the hPR-P4 complex the resulting sum was 101 (Figure 4). However, in hPRDHP, the summarized hydrogen-bond populations were only 85 . In the elePR complexes with both P4 and DHP these values were much more similar with 90 and 95 , respectively. From our data we can conclude that the altered A-ring position in the hPR-DHP complex affected the hydrogen bond network of the binding pocket, both reducing the probability of H-binding via Arg766 as well as destabilizing the intra-residual H-bonds of the binding pocket.Human and Elephant PR LBD Differ in 25033180 DHP PositioningTo identify structural differences in the binding of progesterone and DHP to human and elephant PR, we performed unrestrained molecular dynamics simulations based on all four receptor-ligand combinations. This allowed us to study ligand positioning as well as local receptor flexibilities. The closest-to-average structures of the trajectories in comparison to the X-ray structure are shown in Figure 3. While progesterone docked in a position similar to the Xray structure in both receptors (Figure 3A), DHP exhibited a slightly different orientation compared to the ligand in the crystalstructure (Figure 3B). The binding pocket is relatively restricted around the A-ring, leading to similar positions of the A-rings of both ligands. The ring geometries, however, differ between progesterone and DHP because of the double versus single bond between C4 and C5 (Figure 1). This causes a twist in the ligand leading to different positions of the remaining three rings of progesterone and DHP (seen best Figure 3C, center panels). Yet, the acyl group attached to the D-ring is found in a similar position in all four investigated complexes. Due to the altered geometry of the A-ring, there is no contact between C19 and the receptor. Therefore, DHP bound to hPR adopts a position with the A-ring that is closer to the H3 helix resulting in an A-ring orientation that is different from progesterone (Figure 3B). In the elePR-DHP complex this orientation is not possible, as the methyl side chain of Ala722 would clash with C1 of the ligand. As a result, the DHP Aring in the complex with elePR is shifted away from the H3 helix, adopting an orientation that is similar to that of the progesterone A-ring.Additional Amino Acid Substitutions Increase Ligand Affinities of the PR Accompanied by a Loss of Flexibility of 1326631 the Binding PocketWe next sought to analyze the molecular basis of the overall gain of ligand affinity that we have observed for elePR compared to hPR, by focusing on the additional amino acid differences between both species. Three amino acid exchanges were found in close vicinity to the residues involved in steroid binding: V698M,Elephant Progestin ReceptorElephant Progestin ReceptorFigure 3. In elePR alanine 722 orientates the DHP A-ring into a position similar to progesterone. (A,B) Structures of progesterone (P4) and DHP bound to the human and elephant PR LBD compared to the X-ray structure (PDB code 1a28) (green) generated by molecular-dynamics simulations. The frames of each trajectory were fitted on the start frame using the Ca atoms of the helical parts and resulting averaged coordinates were used for a further fit. Represented are the frames with the least relative deviation to the averaged coordinates. The contour lines represent the solvent/ligand-accessible parts of residue 722. (A) hPR-P4 (orange), elePR-P4 (violet). (B) hPR-DHP (yellow), elePR-DHP.