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, 291 (16), 8795-804

An Intrinsically Disordered Motif Mediates Diverse Actions of Monomeric C-reactive Protein

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An Intrinsically Disordered Motif Mediates Diverse Actions of Monomeric C-reactive Protein

Hai-Yun Li et al. J Biol Chem.

Abstract

Most proinflammatory actions of C-reactive protein (CRP) are only expressed following dissociation of its native pentameric assembly into monomeric form (mCRP). However, little is known about what underlies the greatly enhanced activities of mCRP. Here we show that a single sequence motif, i.e. cholesterol binding sequence (CBS; a.a. 35-47), is responsible for mediating the interactions of mCRP with diverse ligands. The binding of mCRP to lipoprotein component ApoB, to complement component C1q, to extracellular matrix components fibronectin and collagen, to blood coagulation component fibrinogen, and to membrane lipid component cholesterol, are all found to be markedly inhibited by the synthetic CBS peptide but not by other CRP sequences tested. Likewise, mutating CBS in mCRP also greatly impairs these interactions. Functional experiments further reveal that CBS peptide significantly reduces the effects of mCRP on activation of endothelial cells in vitro and on acute induction of IL-6 in mice. The potency and specificity of CBS are critically determined by the N-terminal residues Cys-36, Leu-37, and His-38; while the versatility of CBS appears to originate from its intrinsically disordered conformation polymorphism. Together, these data unexpectedly identify CBS as the major recognition site of mCRP and suggest that this motif may be exploited to tune the proinflammatory actions of mCRP.

Keywords: C-reactive protein; atherosclerosis; endothelial dysfunction; extracellular matrix; inflammation; intrinsically disordered region; protein motif.

Figures

FIGURE 1.
FIGURE 1.
An overview of examined CRP peptides. A, experiment design. The physical properties of synthesized peptides computed by ProtParam online tool (B) and their reported bioactivities (C) are also indicated.
FIGURE 2.
FIGURE 2.
CBS peptide inhibits the binding of mCRP to various types of ligands. ApoB (A), C1q (B), fibronectin (C), collagen IV (D), fibrinogen (E) in TBS or cholesterol (F) in ethanol was immobilized onto microtiter wells (n = 3–4). mCRP at different concentrations was added and binding was evaluated using mCRP-specific mAb 3H12 (left panel). The half-saturated binding concentration of mCRP was selected to test the inhibitory effects of co-added peptides (middle panel). The peptide of CBS (a.a. 35–47) emerged as the most potent inhibitor against all interactions. The IC50 of CBS or CBS in multiple antigenic peptide (MAP) format with 4 branches was further determined (right panel).
FIGURE 3.
FIGURE 3.
CBS exhibits intrinsically disordered region-like conformation features. A, crystal structure of subunit A of pentameric CRP (PDB 1B09) in which a.a. 35–47 is highlighted. B, circular dichroism spectra of CBS peptide. Estimated contents of secondary structure elements are indicated. C–E, structures of CBS, a.a. 92–106 and a.a. 107–118 were taken from the crystal structure (PDB 1B09) and subjected to a 100-ns molecular dynamics simulation. C, frequency of each residue in the indicated peptide exists as β-sheet, α-helix or coil conformations across the 100-ns simulation. D, backbone root-mean-squared deviation (RMSD) from the initial crystal structure was plotted with time. E, conformation snapshots at 0, 5, 50, 62.5, 80, and 100 ns of simulation. CBS fluctuated through a range of conformations with preserved N-terminal β-strand and C-terminal loop. These were in contrast to the rather stable conformations of control CRP peptides during the simulation.
FIGURE 4.
FIGURE 4.
The N- and C-terminal contribute differentially to the inhibitory capacity of CBS peptide. CBS mutants with truncations in its N or C terminus were tested for their effects on mCRP binding to ApoB (A), C1q (B), fibronectin (C), collagen IV (D), fibrinogen (E), and insertion into the cholesterol-enriched monolayers (F) (n = 3). In A–E, assays were performed as described in Fig. 2. In F, monolayer technique (see “Experimental Procedures”) was used to more appropriately model the interaction of mCRP with cholesterol in a membraneous environment. In most cases, the inhibitory effects of CBS were completely lost following N-terminal truncation, but was not abrogated by C-terminal truncation.
FIGURE 5.
FIGURE 5.
The 3 N-terminal residues determine the ligand specificity of CBS. CBS mutants with point mutations at the indicated N-terminal residues were tested for their effects on mCRP binding to ApoB (A), C1q (B), fibronectin (C), collagen IV (D), fibrinogen (E), and insertion into the cholesterol-enriched monolayers (F) (n = 3). The inhibition of mCRP binding to distinct ligands depend on different combinations of N-terminal residues.
FIGURE 6.
FIGURE 6.
Mutating CBS in mCRP impairs ligand binding. mCRP wild type or mutants with altered CBS were expressed in E. coli and purified to test their binding to ApoB (A), C1q (B), fibronectin (C), collagen IV (D), fibrinogen (E), and cholesterol (F) (n = 4). The binding of mCRP mutants was greatly impaired or nearly abrogated by mutating the 3 N-terminal residues (C36A/L37A/H38A) or deleting CBS (Δ35–47).
FIGURE 7.
FIGURE 7.
CBS inhibits mCRP-induced cell adhesion. A, confluent HAEC monolayers were pre-incubated with 0.5 mg/ml CBS or scrambled CBS followed by stimulation with 50 μg/ml Cys-mutated mCRP. Treatment with 1 μg/ml LPS was used as the positive control. Adhesion molecules on the surfaces of HAECs were evaluated by flow cytometry using PE-labeled anti-E-selectin, APC-labeled anti-ICAM-1, and FITC-labeled anti-VCAM-1 mAbs (n = 3). B, confluent monolayers of Calcein AM-labeled HAECs were pre-incubated with 2.5 mg/ml CBS or control peptide (a.a. 1–16) for 30 min. Following gentle washing, Hoechst 33342-labeled U937 cells were added together with 50 μg/ml Cys-mutated mCRP for 60 min. U397 adhesion was visualized by confocal microscopy. In control experiments, peptide pre-treatment and mCRP stimulation were omitted. C, quantification of experiments in B (n = 9). ***, p < 0.001; **, p < 0.01. D, mCRP at the indicated concentrations was immobilized onto microtiter wells overnight at 4 °C. After blocking with 0.1% BSA, U937 cells were added with 2.5 mg/ml CBS or control peptide (a.a. 1–16) for 60 min followed by gentle rinsing and fixation. U937 adhesion was quantified by crystal violet staining (n = 3–4).
FIGURE 8.
FIGURE 8.
CBS inhibits mCRP-induced peritoneal IL-6 release. Male Kunming mice were intraperitoneally injected with PBS or Cys-mutated mCRP (2.5 mg/kg) with vehicle, CBS, scrambled CBS or a control peptide (a.a. 27–38) (14 mg/kg). Peritoneal fluid was collected 2 h later and assayed for IL-6. ***, p < 0.001.
FIGURE 9.
FIGURE 9.
Schematic illustration of CBS-mediated ligand binding of mCRP. The N-terminal residues in β conformation likely constitute the major interface for ligand binding, whereas the intrinsically disordered C-terminal residues may contribute to the optimal recognition of ligands with distinct topologies by dynamical sampling of appropriate spatial configurations.

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