title: Identification of the estrogen receptor Cd-binding sites by chemical modification.
abstract: The widely reported interactions of the estrogen receptor (ER) with endocrine disrupting chemicals (EDCs) present in the environment gave raise to public concern and led to a number of screening and testing initiatives on the international level. Recent studies indicated that certain heavy metals, including cadmium, can mimic the effects of the endogenous estrogen receptor agonist 17beta-estradiol, and lead to estrogen receptor activation. Previous studies of the chimeric proteins, which incorporate the ligand-binding domain of the human ER, identified Cys 381, Cys 447, Glu 523, His 524 and Asp 538 as possible sites of interactions with cadmium. In the present study we utilized the rainbow trout ER ligand-binding domain fused to glutathione-S-transferase, and used Cd-shielding against various types of chemical modification of the fusion protein to study non-covalent interactions between the ER and Cd. The distribution of exposed and shielded residues allowed to identify amino acid residues involved in the interaction. Our data indicated preferential protection of Cys groups by cadmium, suggesting their involvement in the interaction. This supports data found in the literature on the strong binding affinity of the thiol group towards metals. However, not all Cys in the fusion protein sequence were protected against chemical modification, illustrating the importance of their chemical environment. In general, the location of rtER-LBD Cys residues implicated in Cd interactions did not confirm assignments made by alanine-scanning mutagenesis for the hER, probably due to differences in experimental setup and fusion proteins used. The involvement of other functional groups such as carboxylic acids in the Cd interactions, though not confirmed, can not be completely ruled out due to the general limitations of the chemical modification approach discussed in detail. Suggestions for an improved experimental setup were made.
authors.affiliation: Department of Environmental Toxicology, Swiss Federal Institute for Environmental Science and Technology, 8600 Dubendorf, Switzerland.
authors.forename: Victor J
authors.initials: VJ
authors.lastname: Nesatyy
authors.forename: Barbara V
authors.initials: BV
authors.lastname: Rutishauser
authors.forename: Rik I L
authors.initials: RI
authors.lastname: Eggen
authors.forename: Marc J-F
authors.initials: MJ
authors.lastname: Suter
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002104:Cadmium / Q000737:chemistry*; D047628:Estrogen Receptor alpha / Q000737:chemistry / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D016415:Sequence Alignment; D014337:Trout
chemical_list: D047628:Estrogen Receptor alpha; D002104:Cadmium