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.
Mullenix
M C
MC
Department of Microbiology, Ohio State University, Columbus 43210.
Mortensen
R F
RF
D000494:Allosteric Regulation; D000595:Amino Acid Sequence; D001665:Binding Sites; D002097:C-Reactive Protein / Q000737:chemistry / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism*; D002942:Circular Dichroism; D003956:Dialysis; D006801:Humans; D037102:Lectins / Q000378:metabolism; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000378:metabolism; D010767:Phosphorylcholine / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation
D037102:Lectins; D010446:Peptide Fragments; D010767:Phosphorylcholine; D002097:C-Reactive Protein; D002118:Calcium