Delineation of lipopolysaccharide (LPS)-binding sites on hemoglobin: from in silico predictions to biophysical characterization

Abstract

Hemoglobin (Hb) functions as a frontline defense molecule during infection by hemolytic microbes. Binding to LPS induces structural changes in cell-free Hb, which activates the redox activity of the protein for the generation of microbicidal free radicals. Although the interaction between Hb and LPS has implications for innate immune defense, the precise LPS-interaction sites on Hb remain unknown. Using surface plasmon resonance, we found that both the Hb α and β subunits possess high affinity LPS-binding sites, with K(D) in the nanomolar range. In silico analysis of Hb including phospho-group binding site prediction, structure-based sequence comparison, and docking to model the protein-ligand interactions showed that Hb possesses evolutionarily conserved surface cationic patches that could function as potential LPS-binding sites. Synthetic Hb peptides harboring predicted LPS-binding sites served to validate the computational predictions. Surface plasmon resonance analysis differentiated LPS-binding peptides from non-binders. Binding of the peptides to lipid A was further substantiated by a fluorescent probe displacement assay. The LPS-binding peptides effectively neutralized the endotoxicity of LPS in vitro. Additionally, peptide B59 spanning residues 59-95 of Hbβ attached to the surface of Gram-negative bacteria as shown by flow cytometry and visualized by immunogold-labeled scanning electron microscopy. Site-directed mutagenesis of the Hb subunits further confirmed the function of the predicted residues in binding to LPS. In summary, the integration of computational predictions and biophysical characterization has enabled delineation of multiple LPS-binding hot spots on the Hb molecule.

FIGURE 1.

Schematic representation of the structure of LPS (38). GlcN, glucosamine; P, phosphate; KDO, 2-keto-3-deoxyoctulosonic acid; Hep, d-glycero-d-manno-heptose; Gal, galactose; Glc, glucose; NGa, N-acetyl galactosamine; NGc, N-acetyl glucosamine.

FIGURE 2.

Hb subunits possess high affinity for bacterial LPS. S. minnesota LPS was immobilized on an HPA sensor chip. rHbα and rHbβ at 0.1, 0.25, 0.5, and 0.75 μm in HBS were individually injected over the ligand-immobilized chip surface at a flow rate of 30 μl/min. Data were fitted to a 1:1 Langmuir binding model using the separate fit module of the BIAevaluation software (version 4.1). Sensorgrams for the interaction of rHbα (A) and rHbβ (B) with LPS are shown in black. The red lines represent the corresponding fits. Plots of residuals corresponding to the differences between the experimental and best-fit curves are shown in the lower panel. RU, response units. C, kinetic parameters for binding of rHbα and rHbβ to the various ligands.

FIGURE 3.

Computational prediction of LPS-binding sites on Hb. A, prediction of phospho-group binding patches. An algorithm for computing phospho-group binding propensity was applied on human Hb (Protein Data Bank code 1HGB). FhuA protein in complex with LPS (Protein Data Bank code 1QFG) and MD2 protein with bound lipid A (Protein Data Bank code 2E59) served as positive controls. The binding propensity is represented by surface coloring, which varies linearly from white to blue over favorable propensity values from 1 to 30, and from red to white over the unfavorable propensity values from −1 to 0. The ligands, LPS and lipid A, are shown in a licorice representation, with the phosphate atoms colored green. The phosphate atoms of the ligands in FhuA and MD2 are outlined in yellow. Insets show magnified images of the phospho-groups of the ligand along with the surface coloring for the proteins. B, structure-based sequence alignment of vertebrate Hbα chains. Vertebrate Hb proteins were aligned on the basis of structure using the MultiSeq tool of VMD. The alignment for Hbα is shown here. Residues are color-shaded on the basis of predicted propensity of phospho-residue contact in a decreasing order from green to red to blue. Residues with no propensity of contact are indicated in black. Buried residues are indicated in yellow. The helix designations, A–F, for Hb (according to Kendrew's nomenclature (39)) are shown above the alignment. C, molecular docking of the ligand to the predicted LPS-binding sites. The diglucosamine head group of lipid A (1,4′-bisphospho-β-(1,6)-2,2′-N-acetyl-3,3′-O-acetyl-d-glucosamine disaccharide) was used for docking analysis to the predicted LPS-binding sites. Panels I–VIII, top ranking docked poses of the ligand to the predicted LPS-binding sites. Dockings were performed using GLIDE (version 5.0; Schrodinger, LLC, 2007). The residues participating in hydrogen bonding with the ligand and the computed binding energies are indicated. The α subunits are colored gold, and the β subunits are colored violet. The ligand is in stick representation, whereas the polypeptide chains are shown as ribbons. The hydrogen bonds are indicated as dashed lines.

FIGURE 4.

Hb peptides used for empirical validations. A, designations and sequences of the Hb peptides. Nine peptides were designed to incorporate the putative LPS-binding residues (underlined in the sequence). The peptides are designated A and B corresponding to the Hb α and β subunits, respectively. Two additional control peptides were (i) A111, composed of the stretch of residues which were predicted to be non-LPS binding, and (ii) B59sm, mutant peptide obtained by replacing the predicted LPS-binding residues of B59s with aspartic acid. B, mapping of the Hb peptides on the tetrameric structure. Tetrameric Hb showing the peptides designed to incorporate the predicted LPS-binding sites. The α subunits are colored gold, and the β subunits are colored violet. The heme moiety is represented by sticks, whereas the polypeptide chains are shown as ribbons. The peptides are labeled for chains A and B. C, secondary structure of Hb peptides. Far-UV CD spectra of Hb peptides in PBS, pH 7.4, at 298 K. Peptide concentration was 40 μm. A negative peak near 200 nm represents a random coil structure. Panel I, α subunit peptides: A1 (green), A16 (blue), A56 (red), A81 (aqua), A131 (brown), A111 (cyan); and panel II, β subunit peptides: B1 (green), B59 (aqua), B59s (red), B139 (blue), B59sm (black).

FIGURE 5.

Surface plasmon resonance measurements of the interaction of Hb peptides with LPS. Peptides in HBS were injected over the immobilized ligands at a flow rate of 30 μl/min. Sample concentrations were 50, 100, 150, and 200 μm (except for B59, for which the concentrations were 0.01, 0.05, 0.1, and 0.5 μm). The association and dissociation phases of data were separately fitted to a 1:1 Langmuir binding model in the BIAevaluation software (version 4.1). A–F, sensorgrams for the interaction of the peptides with LPS are shown in black. The red lines represent the corresponding fits. Plots of residuals corresponding to the differences between the experimental and best-fit curves are shown in the lower panel. RU, response units. G, representative sensorgrams of the peptides that did not bind LPS at a concentration of 200 μm: A1 (brown), A16 (green), A111 (black), B59sm (red), and B139 (purple). The response indicates a bulk shift without any binding activity. H, kinetic parameters for interaction of peptides with the various ligands.

FIGURE 6.

DC displacement assay for interaction of Hb peptides with lipid A in solution. The occupancy of DC (50 μm) on lipid A (20 μg/ml) was measured in the presence of 0, 0.1, 1, 10, 50, and 100 μm of the various peptides at pH 7.4 in HBS. The fluorescence intensity was measured by excitation at 340 nm and emission at 560 nm. The occupancy was calculated using the formula: occupancy = (F_P − F_D)/(F_L − F_D), where F_D is the fluorescence intensity of DC in the absence of lipid A, F_L is the fluorescence intensity of DC in the presence of lipid A; and F_P is the fluorescence intensity of the solution of DC and lipid A upon the addition of the different concentrations of the peptides. Occupancy is plotted against the log-peptide concentrations for peptides that bind LPS (A) and do not bind LPS (B). Results are the means ± S.D. of three independent experiments done in triplicate.

FIGURE 7.

Neutralization of LPS endotoxicity by Hb peptides. S. minnesota LPS was treated with 1, 10, and 100 μm concentrations of peptides. The endotoxicity of the solutions was measured using rFC-based PyroGene assay. The extent of neutralization of LPS by the peptides is plotted as a percentage of the “LPS only” control for the LPS-binding peptides (A) and non LPS-binding peptides (B). Results are the means ± S.D. of three independent experiments done in triplicate.

FIGURE 8.

LPS-binding peptide, B59, associates with Gram-negative bacteria. A, the binding of B59 to GNB was analyzed by flow cytometry. E. coli and P. aeruginosa were treated with 1 mm B59 for 30 min, with (white) or without (stripes) pre-treatment with 100 ng/ml LPS. The peptide bound to the bacterial surface was detected using primary rabbit anti-Hb antibody (1:400) and secondary phycoerythrin-conjugated goat anti-rabbit IgG (1:200). The fluorescence intensity is plotted in log-fluorescence units versus counts. The negative control bacteria incubated without the peptide are indicated in gray. The results are representative of three independent experiments. B, the association of B59 to the bacterial surface was visualized using scanning electron microscopy coupled with immunogold labeling. Specific binding of the peptide is demonstrated by the colloidal gold (arrows) observed under backscattered electron (BSE) mode. Untreated bacteria served as negative control. Magnification is 15,000×. Bar, 1 μm.

FIGURE 9.

LPS-binding activities of wild-type and mutant Hb subunits. ELISA was performed for quantifying the LPS-binding activity of the mutant Hb subunits: Hb α (A), and Hb β (B). 96-Well Maxisorp^TM microtiter plates were coated with S. minnesota LPS. The unbound sites were blocked with 2% BSA, and increasing concentrations of the recombinant proteins were added to each well. Anti-Hb antibody was added followed by horseradish peroxidase-linked secondary antibody. The peroxidase enzyme activity was determined at 405 nm. Results are the means ± S.D. of three independent experiments done in triplicate.