Probing the C1q-binding site on human C-reactive protein by site-directed mutagenesis.
We have used oligonucleotide-directed site-specific mutagenesis to investigate structural determinants of the C1q-binding site of C-reactive protein (CRP). Eleven mutant rCRP cDNAs, D112N; D112A; D112K; D112E; K114T; K114A; K114E; K114R; R116L; D112N, K114T; and D112N, R116L were constructed and expressed in COS cells. Wild-type (wt) and all mutant rCRPs bound to phosphocholine-substituted BSA and also to pneumococcal C-polysaccharide with apparent avidities similar to native CRP, except for the R116L mutant which bound slightly less avidly. Substitution of Asn, Ala, or Lys for Asp-112 resulted in decreased avidity of ligand-bound CRP for C1q and also in decreased C-activating efficiency as estimated from a C3-fragment deposition assay. However, complexes of the D112E mutant reproducibly bound better to C1q and activated the classical pathway more efficiently than wt rCRP. Substitution of Thr, Ala, or Glu for Lys-114 increased the avidity for C1q by 2- to 3-fold and the efficiency of classical pathway activation by 20- to 30-fold compared with wt CRP. In contrast, the K114R mutant was only slightly different from wt CRP. Substitution of Leu for Arg-116 did not significantly affect C1q-binding but resulted in increased C-activating efficiency. The data indicate that the negative charge of residue Asp-112 plays a major role in the formation of the C1q-binding site of CRP and that the positively-charged residue Lys-114 and to a lesser extent also Arg-116 play important but indirect roles in C1q-binding and activation of C by CRP complexes.
Agrawal
A
A
Department of Medicine, University of Alabama at Birmingham, AL 35294.
Volanakis
J E
JE
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D002097:C-Reactive Protein / Q000378:metabolism*; D015922:Complement C1q / Q000378:metabolism*; D003176:Complement C3 / Q000378:metabolism; D006801:Humans; D007074:Immunoglobulin G / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D013329:Structure-Activity Relationship
D003176:Complement C3; D007074:Immunoglobulin G; D015922:Complement C1q; D002097:C-Reactive Protein