Characterization of the Estradiol-Binding Site Structure of Human Protein Disulfide Isomerase (PDI)

BACKGROUND: Earlier studies showed that 17β-estradiol (E(2)), an endogenous female sex hormone, can bind to human protein disulfide isomerase (PDI), a protein folding catalyst for disulfide bond formation and rearrangement. This binding interaction can modulate the intracellular levels of E(2) and its biological actions. However, the structure of PDI's E(2)-binding site is still unclear at present, which is the focus of this study. METHODOLOGY/PRINCIPAL FINDINGS: The E(2)-binding site structure of human PDI was studied by using various biochemical approaches coupled with radiometric receptor-binding assays, site-directed mutagenesis, and molecular computational modeling. Analysis of various PDI protein fragments showed that the [(3)H]E(2)-binding activity is not associated with the single b or b' domain but is associated with the b-b' domain combination. Computational docking analyses predicted that the E(2)-binding site is located in a hydrophobic pocket composed mainly of the b' domain and partially of the b domain. A hydrogen bond, formed between the 3-hydroxyl group of E(2) and His256 of PDI is critical for the binding interaction. This binding model was jointly confirmed by a series of detailed experiments, including site-directed mutagenesis of the His256 residue coupled with selective modifications of the ligand structures to alter the binding interaction. CONCLUSIONS/SIGNIFICANCE: The results of this study elucidated the structural basis for the PDI–E(2) binding interaction and the reservoir role of PDI in modulating the intracellular E(2) levels. The identified PDI E(2)-binding site is quite different from its known peptide binding sites. Given that PDI is a potential therapeutic target for cancer chemotherapy and HIV prevention and that E(2) can inhibit PDI activity in vitro, the E(2)-binding site structure of human PDI determined here offers structural insights which may aid in the rational design of novel PDI inhibitors.