SITE-DIRECTED MUTAGENESIS OF THE PHOSPHOCHOLINE-BINDING SITE OF HUMANC-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 constructe d an additional mutant, T76Y, to probe the structural determinants of the PCh-binding site of CRP. Binding properties of four mutant CRPs, K 57Q/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-conjugat ed agarose columns, Purified mutant CRPs, K57Q/R58G/W67K and T76Y fail ed to bind to solid phase, PCh-substituted BSA. They did, however, bin d to immobilized PnC, although with substantially decreased avidity co mpared with wt CRP. W67K, K57Q/R58G/W67K, and T76Y CRP required a 10-f old higher Ca2+ concentration than wt CRP to bind PnC and exhibited de creased avidity for mAb EA4.1, which recognizes a Ca2+-dependent epito pe. We conclude that Thr(76) is a determinant of the PCh-binding site, probably interacting with the choline group, This conclusion is suppo rted by recent crystallographic data indicating that this residue part icipates in the formation of a hydrophobic pocket that constitutes the binding site for choline, Trp(67), LyS(57), and Arg(58) do not direct ly contact PCh, but appear to be required for the proper conformation of the binding site.