Site-directed mutagenesis of the phosphocholine-binding site of human C-reactive protein: role of Thr76 and Trp67.
We have reported previously that residues Lys57, Arg58, and Trp67 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 Thr76 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. Trp67, Lys57, and Arg58 do not directly contact PCh, but appear to be required for the proper conformation of the binding site.
Agrawal
A
A
Division of Clinical Immunology and Rheumatology, Department of Medicine, University of Alabama at Birmingham 35294, USA.
Lee
S
S
Carson
M
M
Narayana
S V
SV
Greenhough
T J
TJ
Volanakis
J E
JE
D002097:C-Reactive Protein / Q000235:genetics*; D006801:Humans; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010767:Phosphorylcholine / Q000378:metabolism*; D011485:Protein Binding / Q000235:genetics / Q000276:immunology; D000683:Serum Amyloid P-Component / Q000378:metabolism; D013912:Threonine / Q000032:analysis; D014364:Tryptophan / Q000032:analysis
D000683:Serum Amyloid P-Component; D010767:Phosphorylcholine; D013912:Threonine; D014364:Tryptophan; D002097:C-Reactive Protein