Molecular interfaces of the galactose-binding protein Tectonin domains in host-pathogen interaction

Abstract

Beta-propeller proteins function in catalysis, protein-protein interaction, cell cycle regulation, and innate immunity. The galactose-binding protein (GBP) from the plasma of the horseshoe crab, Carcinoscorpius rotundicauda, is a beta-propeller protein that functions in antimicrobial defense. Studies have shown that upon binding to Gram-negative bacterial lipopolysaccharide (LPS), GBP interacts with C-reactive protein (CRP) to form a pathogen-recognition complex, which helps to eliminate invading microbes. However, the molecular basis of interactions between GBP and LPS and how it interplays with CRP remain largely unknown. By homology modeling, we showed that GBP contains six beta-propeller/Tectonin domains. Ligand docking indicated that Tectonin domains 6 to 1 likely contain the LPS binding sites. Protein-protein interaction studies demonstrated that Tectonin domain 4 interacts most strongly with CRP. Hydrogen-deuterium exchange mass spectrometry mapped distinct sites of GBP that interact with LPS and with CRP, consistent with in silico predictions. Furthermore, infection condition (lowered Ca(2+) level) increases GBP-CRP affinity by 1000-fold. Resupplementing the system with a physiological level of Ca(2+) did not reverse the protein-protein affinity to the basal state, suggesting that the infection-induced complex had undergone irreversible conformational change. We propose that GBP serves as a bridging molecule, participating in molecular interactions, GBP-LPS and GBP-CRP, to form a stable pathogen-recognition complex. The interaction interfaces in these two partners suggest that Tectonin domains can differentiate self/nonself, crucial to frontline defense against infection. In addition, GBP shares architectural and functional homologies to a human protein, hTectonin, suggesting its evolutionarily conservation for approximately 500 million years, from horseshoe crab to human.

FIGURE 1.

GBP tends to exist in oligomeric forms. A, crude plasma and purified GBP were separated by SDS-PAGE with or without reducing agent. Immunoblotting (IB) was performed with anti-GBP antibody. R, reducing condition; NR, nonreducing condition. B, matrix-assisted laser desorption ionization time-of-flight spectra identified the purified 52, 26, and 18 kDa protein bands as the dimer, monomer, and N-terminal fragment of GBP, respectively.

FIGURE 2.

GBP binds sugar moieties of LPS with high affinity. A, chemical structure of LPS. GlcNAc is located on the outer core of LPS. Hep, heptose; P, phosphate; PE, phosphoethanolamine; KDO, 3-deoxy-α-d-manno-octulosonic acid; Ara, arabinose. The outermost sugar residue of each LPS truncate is colored. B–E, ELISA to measure GBP-ligand binding. The GBP ligands (LPS, ReLPS, lipid A, LTA, or GlcNAc-bovine serum albumin (BSA)) were incubated overnight in binding buffer (see supplemental Materials and Methods) on 96-well Polysorp^TM microplates. The unbound sites were blocked with 1% bovine serum albumin, and serially diluted GBP (with or without preincubation with GlcNAc) was added to each well. Anti-GBP antibody was added followed by horseradish peroxidase-linked secondary antibody. The peroxidase enzyme activity was determined at 405 nm. F, SPR-derived binding constants of GBP to LPS, LPS-truncates, or GlcNAc. The apparent K_D values were calculated by using BIAevaluation software version 4.1. Suffix n and i refer to naïve (uninfected) and infected experimental conditions, respectively. G, SPR analysis of GBP binding with LPS, with and without dithiothreitol (DTT) treatment.

FIGURE 3.

Three-dimensional model of GBP. A, sequence alignment of GBP to TL-1 is shown. B, homology model of GBP predicted a 6-bladed β-propeller protein, containing 8 cysteine residues (yellow). C, protein sequence of GBP shows six Tectonin domain repeats with an 8-residue tail. The modeled β-sheets are numbered accordingly. β-Strands (underlined) are predicted by PSIPRED. D, Ramachandran plot shows that the outlier residues remain close to the boundaries of the permitted ψ-ϕ values (light blue contours), indicating a reliably modeled structure. E, GBP is predominantly hydrophilic (blue) with scattered hydrophobic patches (red). The molecule folds into a toroidal structure around a funnel-shaped tunnel with a larger cavity on the top and a smaller crevice at the bottom.

FIGURE 4.

Yeast two-hybrid analysis shows that specific Tectonin domains of GBP interact with CRP. Single, double, and triple GBP Tectonin domain subclones were tested for their interaction with CRP and with full-length GBP. The pGBKT7 vector containing GBP Tectonins domains/full-length GBP and pGADT7 vector containing CRP/full-length GBP were co-transformed and spotted on SD-Leu-Trp plates (double-dropout control) and SD-His-Ade-Leu-Trp (quadruple-dropout). The strength of interaction is indicated as +/−.

FIGURE 5.

Docking and identification of GBP surfaces that bind GlcNAc, lipid A, and/or CRP. A, GlcNAc and lipid A structures used for docking to GBP are shown. B, GlcNAc (orange) was docked to GBP, and binding energies were quantified. Circled numbers correspond to the Tectonin domains. Inset, GlcNAc were docked to the clefts (hydrophobic, red; hydrophilic, blue) between the propeller blades. C, lipid A (fatty acid chains, green; glucosamine, blue; phosphates, red) was docked to GBP. The lipid A molecule overlapped one of the GlcNAc binding sites. D–F, HDMS experiments are shown. D, GBP interaction sites with GlcNAc (blue)/lipid A (yellow). Peptides showing change in deuterium uptake were mapped onto the surface of GBP. GBPⁱ showed an additional peptide (2–13) binding to lipid A (purple). Top docking results (GlcNAc, orange; lipid A, green-blue-red) are included for comparison. E, GBP interaction sites with CRP. Peptides involved in deuterium uptake (red, decrease; green, increase). F, CRP peptides that bind GBP. Surfaces in blue and red both showed decreased deuterium incorporation. The calcium (yellow) binding site on CRP is in close proximity and overlapping with the colored surfaces.

FIGURE 6.

Infection increases the affinity of LPS and CRP to GBP. A–C, SPR analysis of GBP, which was first bound to immobilized lipid A, followed by CRP. GBPⁿ-CRPⁿ showed apparent K_D of 2.10 × 10⁻⁷ m, whereas GBPⁱ-CRPⁱ showed 1000-fold increased affinity (apparent K_D of 1.66 × 10⁻¹⁰ m). Depletion of calcium resulted in a 1000-fold increase in affinity (apparent K_D of 3.10 × 10⁻¹⁰ m) of GBPⁿ-CRPⁿ, similar to that of GBPⁱ-CRPⁱ. D and E, supplementing with 2.5 and 10 mm Ca²⁺ did not return the binding affinity of GBPⁱ-CRPⁱ to the basal state. F, proposed model of interaction and formation of the core pathogen-recognition complex. The GBP Tectonin domains 1 and 6 (green circles) bind lipid A of LPS, which are displayed on the Gram-negative bacterium (gray), whereas Tectonin domain 4 (blue circle) interacts with CRP, as determined by SPR, yeast two-hybrid and HDMS experiments. The pathogen-recognition interactome recruits other PRRs such as carcinolectins, CL5 (12), to further stabilize and form the antimicrobial complex to drive downstream effectors and complement activation pathways.