title: Two mutants of human heparin binding protein (CAP37): toward the understanding of the nature of lipid A/LPS and BPTI binding.
abstract: Heparin binding protein (HBP) is an inactive serine protease homologue with important implications in host defense during infections and inflammations. Two mutants of human HBP, [R23S,F25E]HBP and [G175Q]HBP, have been produced to investigate structure-function relationships of residues in the putative lipid A/lipopolysaccharide (LPS) binding site and BPTI (bovine pancreatic trypsin inhibitor) binding site. The X-ray structures have been determined at 1.9 A resolution for [G175Q]HBP and at 2.5 A resolution for the [R23S,F25E]HBP mutant, and the structures have been fully refined to R-factors of 18.2 % and 20.7 %, respectively. The G175Q mutation does not alter the overall structure of the protein, but the ability to bind BPTI has been eliminated, and the mutant mediates only a limited stimulation of the LPS-induced cytokine release from human monocytes. The lipid A/LPS binding property of [G175Q]HBP is comparable with that of native HBP. The R23S,F25E mutations do not affect the binding of lipid A/LPS and BPTI or the LPS-induced cytokine release from human monocytes. This shows that two diverse ligands, lipid A/LPS and BPTI, do not share binding sites. Previously, there was convincing evidence for the proposed lipid A/LPS binding site of HBP. Unexpectedly, the extensive structural changes introduced by mutation of Arg23 and Phe25 do not affect the binding of lipid A/LPS, indicating that another not yet identified site on HBP is involved in the binding of lipid A/LPS.
authors.affiliation: Department of Medicinal Chemistry, Royal Danish School of Pharmacy, Copenhagen, Denmark. kastrup@medchem.dfh.dk
authors.forename: J S
authors.initials: JS
authors.lastname: Kastrup
authors.forename: V
authors.initials: V
authors.lastname: Linde
authors.forename: A K
authors.initials: AK
authors.lastname: Pedersen
authors.forename: B
authors.initials: B
authors.lastname: Stoffer
authors.forename: L F
authors.initials: LF
authors.lastname: Iversen
authors.forename: I K
authors.initials: IK
authors.lastname: Larsen
authors.forename: P B
authors.initials: PB
authors.lastname: Rasmussen
authors.forename: H J
authors.initials: HJ
authors.lastname: Flodgaard
authors.forename: S E
authors.initials: SE
authors.lastname: Bjørn
mesh_terms: D000818:Animals; D023181:Antimicrobial Cationic Peptides; D007611:Aprotinin / Q000378:metabolism*; D001665:Binding Sites; D001798:Blood Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002417:Cattle; D003460:Crystallization; D006023:Glycoproteins / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D015850:Interleukin-6 / Q000378:metabolism; D007456:Iodine Isotopes; D008050:Lipid A / Q000737:chemistry / Q000378:metabolism*; D008070:Lipopolysaccharides / Q000737:chemistry / Q000378:metabolism*; D009000:Monocytes / Q000378:metabolism; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D014961:X-Ray Diffraction
chemical_list: C062991:AZU1 protein, human; D023181:Antimicrobial Cationic Peptides; D001798:Blood Proteins; D002352:Carrier Proteins; D006023:Glycoproteins; D015850:Interleukin-6; D007456:Iodine Isotopes; D008050:Lipid A; D008070:Lipopolysaccharides; D011994:Recombinant Proteins; D007611:Aprotinin