Site-directed mutagenesis of the phosphocholine-binding site of human C-reactive protein - Role of Thr(76) and Trp(67)

We have reported previously that residues Lys(57), Arg(58), and Trp(67) of human C-reactive protein (CRP) contribute to the structure of the phosphocholine (PCh)-binding site, In this study, based on the three-dimensional structures of human CRP and serum amyloid P, we constructed an additional mutant, T76Y, to probe the structural determinants of the PCh-binding site of CRP. Binding properties of four mutant CRPs, K57Q/R58G, W67K, K57Q/R58G/W67K, and T76Y were compared. Wild-type (wt) and all mutant CRPs were purified by affinity chromatography on PCh-, pneumococcal C-polysaccharide (PnC)-, or phosphoethanolamine-conjugated agarose columns, Purified mutant CRPs, K57Q/R58G/W67K and T76Y failed to bind to solid phase, PCh-substituted BSA. They did, however, bind to immobilized PnC, although with substantially decreased avidity compared with wt CRP. W67K, K57Q/R58G/W67K, and T76Y CRP required a 10-fold higher Ca2+ concentration than wt CRP to bind PnC and exhibited decreased avidity for mAb EA4.1, which recognizes a Ca2+-dependent epitope. We conclude that Thr(76) is a determinant of the PCh-binding site, probably interacting with the choline group, This conclusion is supported by recent crystallographic data indicating that this residue participates in the formation of a hydrophobic pocket that constitutes the binding site for choline, Trp(67), LyS(57), and Arg(58) do not directly contact PCh, but appear to be required for the proper conformation of the binding site.