Role of aspartate 351 in transactivation and active conformation of estrogen receptor α

Abstract

Estrogen-dependent transcriptional activation by estrogen receptor (ER ) depends on the conformation of helices 3 and 12 in the ligand-binding domain. To better understand the function of helix 3 in ER , we examined the role of charged residues, which are conserved in most steroid receptors in helix 3, in estrogen-dependent transactivation. The replacement of Asp-351 with lysine (D351K) or leucine (D351 L) completely abolished estrogen-dependent transactivation without affecting estrogen-binding, DNA-binding and homodimerization activities in ER . The mutations dramatically reduced the ligand-dependent activation function 2 activity and impaired the ability of ER to bind p160 coactivators. In addition, the D351K mutant effectively inhibited the transcriptional activation activity of wild-type ER . Furthermore Asp-351 was required not only for the estrogen-dependent conformational change of wild-type ER but also for the constitutive transcriptional activity and ligand-independent active conformation of ER mutant Y537N. Similarly, in the orphan nuclear receptor called estrogen-related receptor 3 (ERR3), the replacement of Asp-273 (the corresponding amino acid to Asp-351 in ER ) with lysine abolished constitutive transcriptional activity of ERR3 without affecting DNA-binding activity and impaired the ability of the receptor to interact with p160 coactivators. These data suggest a role of Asp-351 in inducing and stabilizing the active conformation of ER , and our results experimentally confirm the concept that Asp-351 in helix 3 interacts with the amide hydrogen of L540 in helix 12 to form a transcriptionally competent surface for binding p160 coactivators.

Structural and chemical basis for enhanced affinity and potency for a large series of estrogen receptor ligands: 2D and 3D QSAR studies

“…The maps indicate that the nitrogen pyrrolidine ring is important for the antagonist biocharacter. This parameter was similar to some crystallographic structures co-complexed with antagonist ligands and some studies, which designate the interactions between N of the heterocycle and ASP351 to be responsible for the antiestrogenic activity [48][49][50][51]. Our results also agree with previous studies, which reported that the optimal length of the link is represented by two carbons [28].…”

Involvement of a Hydrophobic Pocket and Helix 11 in Determining the Modes of Action of Prenylated Flavonoids and Isoflavonoids in the Human Estrogen Receptor

“…[30] Unfortunately,n oc rystal structure in which helix 3i s displaced is available to confirmthis hypothesis.…”

Synthesis and evaluation of 11β-(4-Substituted phenyl) estradiol analogs: Transition from estrogen receptor agonists to antagonists

“…It has been observed previously that replacement of Asp-351 by neutral amino acids did not significantly affect the binding affinity of antagonists but did influence the subsequent recruitment of coregulatory peptides. 81 Weatherman and co-workers, noted in their study of GW7604 derivatives, in which a terminal carboxy group of a nonsteroidal antiestrogen was replaced by an amido or methyl ketone moiety, that affinity but not efficacy was affected. 82 Further extension of the 4-substituent, as seen in RU58668 18 , or the ω-substituted triethyleneglycol derivatives 12 and 14, in which the beta atom is carbon or oxygen gave equally potent ER ligands with roughly comparable anti-estrogenic properties.…”