title: Exposing a hidden functional site of C-reactive protein by site-directed mutagenesis.
abstract: C-reactive protein (CRP) is a cyclic pentameric protein whose major binding specificity, at physiological pH, is for substances bearing exposed phosphocholine moieties. Another pentameric form of CRP, which exists at acidic pH, displays binding activity for oxidized LDL (ox-LDL). The ox-LDL-binding site in CRP, which is hidden at physiological pH, is exposed by acidic pH-induced structural changes in pentameric CRP. The aim of this study was to expose the hidden ox-LDL-binding site of CRP by site-directed mutagenesis and to generate a CRP mutant that can bind to ox-LDL without the requirement of acidic pH. Mutation of Glu(42), an amino acid that participates in intersubunit interactions in the CRP pentamer and is buried, to Gln resulted in a CRP mutant (E42Q) that showed significant binding activity for ox-LDL at physiological pH. For maximal binding to ox-LDL, E42Q CRP required a pH much less acidic than that required by wild-type CRP. At any given pH, E42Q CRP was more efficient than wild-type CRP in binding to ox-LDL. Like wild-type CRP, E42Q CRP remained pentameric at acidic pH. Also, E42Q CRP was more efficient than wild-type CRP in binding to several other deposited, conformationally altered proteins. The E42Q CRP mutant provides a tool to investigate the functions of CRP in defined animal models of inflammatory diseases including atherosclerosis because wild-type CRP requires acidic pH to bind to deposited, conformationally altered proteins, including ox-LDL, and available animal models may not have sufficient acidosis or other possible modifiers of the pentameric structure of CRP at the sites of inflammation.
authors.affiliation: Department of Pharmacology, Quillen College of Medicine, East Tennessee State University, Johnson City, Tennessee 37614, USA.
authors.forename: Sanjay K
authors.initials: SK
authors.lastname: Singh
authors.forename: Avinash
authors.initials: A
authors.lastname: Thirumalai
authors.forename: David J
authors.initials: DJ
authors.lastname: Hammond
authors.forename: Michael K
authors.initials: MK
authors.lastname: Pangburn
authors.forename: Vinod K
authors.initials: VK
authors.lastname: Mishra
authors.forename: David A
authors.initials: DA
authors.lastname: Johnson
authors.forename: Antonio E
authors.initials: AE
authors.lastname: Rusiñol
authors.forename: Alok
authors.initials: A
authors.lastname: Agrawal
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites; D002097:C-Reactive Protein / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008077:Lipoproteins, LDL / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D010449:Peptide Mapping; D011485:Protein Binding
chemical_list: D008077:Lipoproteins, LDL; C099252:oxidized low density lipoprotein; D002097:C-Reactive Protein