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 [(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.

Figure 1. Both PDI and its b-b' fragment can bind E₂.

(A). Domain organization of the human PDI protein. The letters A, B and C in boxes that represent the β-α-β, α-β-α, and β-β-α secondary-structure elements, respectively, of the thioredoxin fold, are adopted from an earlier study and were used to guide the design of various PDI protein fragments as shown in this figure and Figure S1. (B) and (D). SDS-PAGE analysis of two histidine-tagged PDI fragments and the full-length protein, which were selectively expressed in E. coli cells (panel B) and then purified using affinity chromatography (panel D). (C) and (E). The binding of [³H]E₂ by either cell lysates (at a final protein concentration of 1 mg/mL; panel C) or by purified proteins (at a final concentration of 0.5 µM; panel E) after incubation with 4.5 nM [³H]E₂ in the absence or presence of 10 µM non-radioactive E₂. (F). SDS-PAGE analysis of selectively-expressed GST-tagged PDI protein fragments in E. coli cells. (G). The binding of [³H]E₂ by cell lysates containing the GST-tagged PDI fragments. For the quantitative data, each value is the mean ± S.D. of triplicate determinations.

Figure 2. Determination of the dissociation constant (K_d) of the full-length human PDI protein (A) and its b-b' fragment (B) for E₂.

Equilibrium analysis was used to determine the dissociation constants as described in the Materials and Methods section. The concentrations of free and total [³H]E₂ (i.e., the sum of PDI-bound [³H]E₂ and free [³H]E₂) were determined by scintillation counting calibrated against standard concentrations of [³H]E₂ (see Figure S2B). Total [³H]E₂ subtracted by free [³H]E₂ gives rise to PDI-bound [³H]E₂. The upper right insets show the binding curves were obtained using curve regression analysis (hyperbola model) of the SigmaPlot software. Each value is the mean of duplicate determinations.

Figure 3. Docking analysis of the binding interaction of E₂ inside human PDI b-b' fragment.

(A). Overview of the docking result of the E₂ binding in the PDI b-b' fragment. E₂ and His256 are shown in the ball-and-stick format and colored according to atoms. The protein structure is shown in ribbon. Yellow colored region denotes the b domain and magenta colored region denotes the b' domain. Secondary structural elements are labeled according the NMR structure of the fragment. (B). A close-up view of the docking result of the E₂-PDI binding mode, showing that a hydrogen bond (by green dash) is formed between the 3-hydroxyl group of E₂ (a hydrogen bond donor) and PDI-His256 (a hydrogen bond acceptor). (C). Interaction of E₂ with the amino acid residues inside the binding pocket. Labeling of amino acid residues is shown in yellow for the b domain and in magenta for the b' domain. E₂ molecule is colored in yellow. Amino acid residues are shown in the ball-and-stick format and colored according to atoms, i.e., green for carbon, red for oxygen, white for hydrogen, and blue for nitrogen. (D). Plots of the docking result of the E₂ binding with PDI b-b' fragment. The distance is in angstroms. E₂ is colored in blue and His256 is in magenta. Note that the amino acid residues of the b and b' domains are colored differently (black for the b domain and red for the b' domain).

Figure 4. H256L mutant protein lacks E₂-binding activity.

(A) and (B). The [³H]E₂ binding by E. coli cell lysates containing the selectively-expressed wild-type or H256L mutant DPI proteins (panel A) or by purified proteins (panel B) after incubation with 4.5 nM [³H]E₂ in the absence or presence of non-radioactive E₂ in excess (10 µM). Inset in panel A shows the SDS-PAGE analysis of the cell lysates. (C). The PDI-bound [³H]E₂ (wild-type and H256L mutant proteins) against increasing concentrations of [³H]E₂ (200 to 3000 nM). Equilibrium analysis was used to determine the binding activity as described in the Materials and Methods section. The concentrations of free and total [³H]E₂ (i.e., the sum of PDI-bound [³H]E₂ and free [³H]E₂) were determined by scintillation counting calibrated against standard concentrations of [³H]E₂ (see Figure S2B).

Figure 5. Relative binding activity of PDI for several E₂ derivatives.

(A). Chemical structures of E₂ and several of its analogs used in this study. (B). Relative binding activity of the purified PDI protein (at 20 µg/mL final concentration, purified from E. coli cells) for [³H]E₂ (4.5 nM) in the absence or presence of non-radioactive 10 µM E₂ or its analogs in sodium phosphate buffer (10 mM, pH 7.4). (C). Docking analysis of the binding modes of E₂ and C1 in the binding pocket of the PDI b-b’ fragment. Protein structure is shown in ribbon and colored in magenta. E₂, C1 and His278 are shown in the ball-and-stick format. C1 and His 256 are colored according to atoms and E₂ colored yellow. Green dashes denote hydrogen bonds. α-Helics and β-sheets are labeled according to Figure S3.

Figure 6. Amino acid sequence alignment showing the overlap and difference between PDI's E₂-binding site and its peptide-binding sites.

(A) Sequence alignment was performed between human PDI and PPIp using Clustal W. The E₂-binding sites of human PDI and PDIp, which are colored in red, are based on the results of this study and our earlier study , respectively, along with the structural information for the peptide-binding sites of PDI determined earlier by others (i.e., the NMR titration analysis of PDI-P1 and PDI-P2 , and the structure analysis of PDI-P3 [33]). The arrow indicates those highly-conserved amino acid residues, namely, His256 in PDI and His278 in PDIp, which are essential for their binding interaction with E₂. Based on an earlier study , the essential amino acid residue His256 in PDI is also involved in binding mastoparan (a peptide substrate) and unfolded RNase A . (B). Mapping the E₂-binding site (left part) and peptide-binding site (right part) in the three-dimensional structure of PDI b-b' fragment. Peptide-binding sites include the combined residues from PDI-P1, PDI-P2, and PDI-P3 as shown in panel A. Binding sites (red) are shown in the ball-and-stick format and the whole protein (white) in a cartoon format.