title: A common site within factor H SCR 7 responsible for binding heparin, C-reactive protein and streptococcal M protein.
abstract: The complement inhibitor factor H (fH) interacts via its seventh short consensus repeat (SCR) domain with multiple ligands including heparin, streptococcal M protein and C-reactive protein (CRP). The aim of this study was to localize the residues in SCR 7 required for these interactions. We initially built a homology model of fH SCR 6-7 using the averaged NMR structures of fH SCR 15-16 and vaccinia control protein SCR 3-4 as templates. Electrostatic potentials of the model's surface demonstrated a co-localization of three clusters of positively charged residues on SCR 7, labeled site A (R369 and K370), site B (R386 and K387) and site C (K392). These residues, localized to the linker region preceding SCR 7 and to the end of a "hypervariable loop" in SCR 7, were systematically replaced with uncharged alanine residues in an fH construct containing SCR 1-7. The resulting proteins were expressed in the methylotrophic yeast, Pichia pastoris. By ELISA analysis we demonstrated: first, that substituting site A inhibited heparin and CRP binding; secondly, that substituting site B inhibited binding to heparin, CRP and M protein; and thirdly, that substituting site C clearly inhibited only heparin binding.
authors.affiliation: Department of Microbiology and Infectious Diseases, Flinders Medical Centre, Adelaide, Australia.
authors.forename: Eleni
authors.initials: E
authors.lastname: Giannakis
authors.forename: T Sakari
authors.initials: TS
authors.lastname: Jokiranta
authors.forename: Dean A
authors.initials: DA
authors.lastname: Male
authors.forename: Shoba
authors.initials: S
authors.lastname: Ranganathan
authors.forename: Rebecca J
authors.initials: RJ
authors.lastname: Ormsby
authors.forename: Vince A
authors.initials: VA
authors.lastname: Fischetti
authors.forename: Carolyn
authors.initials: C
authors.lastname: Mold
authors.forename: David L
authors.initials: DL
authors.lastname: Gordon
mesh_terms: D019943:Amino Acid Substitution; D000942:Antigens, Bacterial; D001425:Bacterial Outer Membrane Proteins / Q000378:metabolism*; D001665:Binding Sites; D002097:C-Reactive Protein / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism*; D017242:Complement Factor H / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006493:Heparin / Q000378:metabolism*; D006801:Humans; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D017434:Protein Structure, Tertiary; D020449:Repetitive Sequences, Amino Acid; D040681:Structural Homology, Protein
chemical_list: D000942:Antigens, Bacterial; D001425:Bacterial Outer Membrane Proteins; C493495:CFH protein, human; D002352:Carrier Proteins; C014689:streptococcal M protein; D017242:Complement Factor H; D006493:Heparin; D002097:C-Reactive Protein