Abstract
Estradiol (E(2)), a female sex hormone, has important biological functions. Human pancreas-specific protein disulfide isomerase (PDIp), a protein folding catalyst, was recently found to be able to bind E(2). Here we report the characterization of its E(2)-binding site by using biochemical methods coupled with molecular modeling tools. Analysis of various truncated PDIp proteins showed that the b-b' fragment contains an intact E(2)-binding site that has the same binding affinity as the full-length PDIp protein, with apparent K(d) values of approximately 170 nM. Computational modeling and docking analyses revealed that the E(2)-binding site in the b-b' fragment is located in a hydrophobic pocket composed mainly of the b' domain and partially of the b domain. The hydrogen bond, formed between the 3-hydroxyl group of E(2) (donor) and PDIp's His278 (acceptor), is indispensable for its binding. By contrast, the 17β-hydroxyl group of E(2) is of negligible importance for E(2) binding. This binding model was jointly confirmed by a series of experiments, such as selective mutation of the binding site amino acid residues and selective modification of the ligand structures.