Probing the phosphocholine-binding site of human C-reactive protein by site-directed mutagenesis.
Human C-reactive protein (CRP) can activate the classical pathway of complement and function as an opsonin only when it is complexed to an appropriate ligand. Most known CRP ligands bind to the phosphocholine (PCh)-binding site of the protein. In the present study, we used oligonucleotide-directed site-specific mutagenesis to investigate structural determinants of the PCh-binding site of CRP. Eight mutant recombinant (r) CRP, Y40F; E42Q; Y40F, E42Q; K57Q; R58G; K57Q, R58G; W67K; and K57Q, R58G, W67K were constructed and expressed in COS cells. Wild-type and all mutant rCRP except for the W67K mutants bound to solid-phase PCh-substituted bovine serum albumin (PCh-BSA) with similar apparent avidities. However, W67K rCRP had decreased avidity for PCh-BSA and the triple mutant, K57Q, R58G, W67K, failed to bind PCh-BSA. Inhibition experiments using PCh and dAMP as inhibitors indicated that both Lys-57 and Arg-58 contribute to PCh binding. They also indicated that Trp-67 provides interactions with the choline group. The Y40F and E42Q mutants were found to have increased avidity for fibronectin compared to wild-type rCRP. We conclude that the residues Lys-57, Arg-58, and Trp-67 contribute to the structure of the PCh-binding site of human CRP. Residues Tyr-40 and Glu-42 do not appear to participate in the formation of the PCh-binding site of CRP, however, they may be located in the vicinity of the fibronectin-binding site of CRP.
Agrawal
A
A
Department of Medicine, University of Alabama, Birmingham 35294.
Xu
Y
Y
Ansardi
D
D
Macon
K J
KJ
Volanakis
J E
JE
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D015153:Blotting, Western; D002097:C-Reactive Protein / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002460:Cell Line; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D008099:Liver / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010767:Phosphorylcholine / Q000378:metabolism*; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014162:Transfection
D011994:Recombinant Proteins; D010767:Phosphorylcholine; D002097:C-Reactive Protein; D004247:DNA