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

ABSTRACT: Earlier studies showed that 17?-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.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.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.

SUBMITTER: Fu XM

PROVIDER: S-EPMC3207843 | biostudies-literature | 2011

REPOSITORIES: biostudies-literature

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

Fu Xin-Miao XM Wang Pan P Zhu Bao Ting BT

PloS one 20111103 11

<h4>Background</h4>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.<h4>Methodology/principal findings</h4>The E(2)-binding si ...[more]

PMID: 22073283

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Project description:Hypoxia-inducible factor-1alpha (HIF-1alpha), a transcription factor, plays a critical role in adaption to hypoxia, which is a major feature of diseases, including cancer. Protein disulfide isomerase (PDI) is up-regulated in numerous cancers and leads to cancer progression. PDI, a member of the TRX superfamily, regulates the transcriptional activities of several transcription factors. To investigate the mechanisms by which PDI affects the function of HIF-1alpha, the overexpression or knockdown of PDI was performed. The overexpression of PDI decreased HIF-1alpha expression in the human hepatocarcinoma cell line, Hep3B, whereas the knockdown of endogenous PDI increased its expression. NH4Cl inhibited the decrease in HIF-1alpha expression by PDI overexpression, suggesting that HIF-1alpha was degraded by the lysosomal pathway. HIF-1alpha is transferred to lysosomal membranes by heat shock cognate 70 kDa protein (HSC70). The knockdown of HSC70 abolished the decrease, and PDI facilitated the interaction between HIF-1alpha and HSC70. HIF-1alpha directly interacted with PDI. PDI exists not only in the endoplasmic reticulum (ER), but also in the cytosol. Hypoxia increased cytosolic PDI. We also investigated changes in the redox state of HIF-1alpha using PEG-maleimide, which binds to thiols synthesized from disulfide bonds by reduction. An up-shift in the HIF-1alpha band by the overexpression of PDI was detected, suggesting that PDI formed disulfide bond in HIF-1alpha. HIF-1alpha oxidized by PDI was not degraded in HSC70-knockdown cells, indicating that the formation of disulfide bond in HIF-1alpha was important for decreases in HIF-1alpha expression. To the best of our knowledge, this is the first study to show the regulation of the expression and redox state of HIF-1alpha by PDI. We also demonstrated that PDI formed disulfide bonds in HIF-1alpha 1-245 aa and decreased its expression. In conclusion, the present results showed that PDI is a novel factor regulating HIF-1alpha through lysosome-dependent degradation by changes in its redox state.

Dataset Information

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

Publications

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

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