Mapping the Binding Areas of Human C-reactive Protein for Phosphorylcholine and Polycationic Compounds: RELATIONSHIP BETWEEN THE TWO TYPES OF BINDING SITES

Reiko T Lee,Isamu Takagahara,Yuan C Lee

doi:10.1074/jbc.m106039200

Reiko T Lee, Isamu Takagahara + Show 1 more

Open Access

https://doi.org/10.1074/jbc.m106039200

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Abstract

We developed a fluorescence-based assay method for determining ligand binding activities of C-reactive protein (CRP) in solution. Using this method, we compared the phosphorylcholine (PC)- and polycation-based binding activities of human CRP. The PC-based binding required calcium, whereas a polycation (e.g. poly-l-lysine) was bound in the presence of either calcium or EDTA, the binding being stronger in the presence of EDTA. The published crystallographic structures of CRP and the CRP.PC complex show it to be a ring-shaped pentamer with a single PC-binding site per subunit facing the same direction. As expected from such a structure, binding affinity of a ligand increased tremendously when multiple PC residues were present on a macromolecular structure. In addition to PC-related structures, certain sugar phosphates (e.g. galactose 6-phosphate) are bound near the PC-binding site, and one of the sugar hydroxyl groups appears to interact with CRP. The best small ligands for the polycationic binding site were Lys-Lys and Lys4. Because of the presence of multiple Lys-Lys sequences, polylysines have tremendously enhanced affinity. Although PC inhibits both PC- and polycation-based binding, none of the amines that inhibit polylysine binding inhibits PC binding, suggesting that the PC and polycationic binding sites do not overlap.

Highlights

The x-ray crystallography of PC1⁄7CRP complex shows that the phosphate group of PC coordinates directly to the bound calcium ions
A sole unliganded oxygen of phosphate can form a second ester bond, which would point toward the solvent space away from the protein surface
We observed that CRP interacts with hydroxylated ligands such as Gal-6-P in the area where the second ester is located, most likely via hydrogen bonding to a hydroxyl group located near the phosphate group

Summary

Methods

Proteins and polylysines were determined by microBCA method (10). Amine-containing compounds including polylysines were determined by 2,4,6-trinitrobenzenesulfonic acid method (11). PC-containing compounds were assayed for organic phosphate content (12), and the aldehydo function was determined by neocuproine method (13). Vicinal glycol was determined by combination of periodate oxidation and 2,4,6-tripyridyl-s-triazine method (14) as follows. Sample (20 ␮l) containing up to 0.1 ␮mol of vicinal glycol was incubated with sodium metaperiodate (10 mM, 10 ␮l) for 0.5 h at 37 °C. The solution was diluted to 1 ml with water, and 2 ml of 2,4,6-tripyridyl-s-triazine solution (14) was added. Absorbance at 593 nm was linearly proportional to the vicinal glycol concentration up to absorbance of 0.8. 1H NMR spectra were recorded with a Bruker AMX 300 spectrometer. Elemental analyses were performed by Galbraith Laboratories, Inc. Elemental analyses were performed by Galbraith Laboratories, Inc. (Knoxville, TN)

Results

Discussion

Conclusion