Compared to other mammals and are therefore not able to serve as functional gestagen. Furthermore, the concentration of progesterone neither changes during the ovarian cycle nor increases during pregnancy, indicating the lack of an endocrine role of progesterone in elephants. Searching for the relevant gestagen in elephants revealed high concentrations of the 5-alpha-reduced progestins 5a-dihydroprogesterone and allopregnanolone, both being synthesized in the corpus luteum of the elephant ovary. Serum levels of DHP show a close correlation with the ovarian cyclicity and remain constantly high from onset of pregnancy until parturition. While the binding capacity in mammals for DHP and allopregnanolone is generally low compared to progesterone, elephants can bind DHP with a similar affinity to progesterone indicating a change in the binding specificity of the PR. The PR belongs to the group of steroid hormone receptors. These also include androgen receptor, estrogen receptor, mineralocorticoid receptor and glucocorticoid receptor, which mediate crucial signals in reproduction, metabolism and salt homeostasis. Steroid hormone receptors are part of the nuclear receptor family, which act as hormone-inducible transcription factors. Upon hormone binding, activated PR is translocated to the nucleus, where it binds to specific cis-elements in the enhancer regions of target genes. DNA-bound PR recruits several cofactor complexes to specifically activate or repress the level of transcription. Apart from its genomic actions, PR has also been shown to be involved in several signaling pathways including the MAPK and PI3K pathways through cross-talk with kinases located in the plasma membrane. Despite the complexity of PR actions in the cell. Steroid hormone receptors are modular proteins consisting of an N-terminal regulatory domain, a centrally-located DNAbinding domain and a C-terminal ligand-binding domain. While the DBD is highly conserved between all steroid hormone receptors at the peptide level, the LBD shares conservation in structure and the amino acids flanking the binding pocket. The specificity of LBDs from different steroid hormone receptors towards their respective ligands is based on hydrogen bond networks, hydrophobic interactions, as well as the CUDC-907 1339928-25-4 steric size and shape of the binding pocket. For instance, steroid hormone receptors which bind ligands with a 3-keto group have a conserved glutamine forming a hydrogen bond with the C3-keto group of the A-ring, while steroid receptors binding ligands with a 3-hydroxy group like the ER contain a glutamate at the corresponding position. An example of how ligand specificity of steroid receptors evolved in detail was given for the GR, which evolved from a more promiscuous ancestral receptor that was activated by the mineralocorticoid aldosterone and more weakly by the glucocorticoid cortisol. Two amino acid exchanges in helix 7 of GR specifically reduced the affinity for aldosterone and thus increased the receptor’s specificity for cortisol. While the first exchange repositioned helix 7 closer to the ligand thereby reducing the affinity to all ligands, the second exchange generated a new hydrogen bond to the 17-hydroxyl group, which is unique to cortisol. Additional substitutions in the GR LBD completed the loss of mineralocorticoid affinity, stabilized the new receptor conformation and generated an epistatic ratchet, which made the receptor’s evolution irreversible. In this study, we address the unique ligand specificity of the elephant PR towards favored binding of DHP at the molecular level. Our approach consists of site-directed mutagenesis in combination with in vitro binding studies and molecular dynamics simulations.