Calcium ion binding regions in C-reactive protein: location and regulation of conformational changes.

C-reactive protein (CRP) is a pentameric acute phase serum protein composed of identical 206 amino acid subunits that associate by non-covalent bonds. The biological activities ascribed to CRP are initiated by binding ligands via the single PC-binding site within each subunit. CRP binding to PC requires a conformational change in the intact pentraxin triggered by the binding of two free Ca2+ ions per subunit. Residues 134-148 of each subunit were previously implicated by indirect measures as one of the Ca(2+)-binding sites. In this study, 45Ca2+ autoradiography revealed that fragments of CRP of 6.5 and 16 kDa generated by proteolysis between residues 146 and 147 bind Ca2+ indicating that a second Ca(2+)-binding site is located within the C-terminal 60 amino acids. Synthetic peptides corresponding to residues 134-148 and 152-176 both bound 45Ca2+ in equilibrium dialysis experiments with a Kd = 5.2 x 10(-4) and 1.7 x 10(-4) M, respectively. The addition of Ca2+ to peptide 152-176 induced a shift in the CD-spectra between 210 and 230 nm. Rabbit anti-peptide 152-176 antibody (Ab) inhibited the availability of an epitope within the PC-binding site of CRP recognized by mAb EA4-1. Reactivity of CRP with both anti-peptide 134-148 mAb and anti-peptide 152-176 Ab enhanced the expression of the PC-binding site epitope. The results suggest that the two distinct Ca(2+)-binding sites within each CRP subunit are composed of residues 134-148 and 152-176 and that these two nearly adjacent sites cooperate to exert an allosteric change in conformation allowing access to the PC-binding site.