Binding site of C-reactive protein on M-ficolin.
The binding abilities of human C-reactive protein (CRP) with the C-terminal fibrinogen-like (FBG) domain and the full-length form of human M-ficolin were investigated by pull-down and zonal affinity chromatography analyses. Pull-down assays using an N-acetyl-D-glucosamine (GlcNAc)-agarose column demonstrated that CRP binds to the trimeric FBG domains, and that the GlcNAc-binding ability of the FBG domain is unaffected by CRP binding. Interestingly, the full-length M-ficolin, comprising the N-terminal collagen-like (COL) and C-terminal FBG domains, displayed lower affinity for CRP, and the monomeric FBG domain showed virtually no binding to CRP, as qualitatively judged by zonal affinity chromatography using a GlcNAc column. These results indicated that CRP binding requires the trimeric form of the FBG domain, and that the presence of the COL domain reduces the interaction between CRP and M-ficolin. In addition, pull-down assays using a histidine-tag affinity column demonstrated that neither the full-length M-ficolin nor the trimeric FBG domains, immobilized through their C-terminal histidine tags, showed any affinity for CRP, indicating that the CRP binding site is located near Ala326 at the C-terminus of M-ficolin, spatially close to a neck region (around Pro115) between the FBG and COL domains. From these findings, we concluded that CRP binding is enhanced by conformational bending at the neck region of M-ficolin, to avoid steric hindrance by the COL domain. Such a situation may be generated by oligomeric M-ficolin binding to surfaces with widely distributed ligands, such as pathogens.
Mitsubishi Kagaku Institute of Life Sciences (MITILS), 11 Minamiooya, Machida, Tokyo 194-8511, Japan.
Michikazu
M
Tanio
Kaori
K
Wakamatsu
Toshiyuki
T
Kohno
D001665:Binding Sites; D002097:C-Reactive Protein / Q000378:metabolism*; D002846:Chromatography, Affinity; D018360:Crystallography, X-Ray; D005944:Glucosamine / Q000378:metabolism; D006639:Histidine / Q000378:metabolism; D006801:Humans; D047468:Immunoprecipitation; D037102:Lectins / Q000737:chemistry / Q000378:metabolism*; D050505:Mutant Proteins / Q000378:metabolism; D009842:Oligopeptides / Q000378:metabolism; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism; D000097604:Ficolins
D002097:C-Reactive Protein; D005944:Glucosamine; D006639:Histidine; D037102:Lectins; D050505:Mutant Proteins; D009842:Oligopeptides; D011994:Recombinant Proteins; D000097604:Ficolins; C471213:His-His-His-His-His-His