Characterization of the estradiol-binding site structure of human protein disulfide isomerase (PDI)

Abstract

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 [ 3H]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. © 2011 Fu et al.