title: Identification of a major heparin-binding site in kallistatin.
abstract: Kallistatin is a heparin-binding serine proteinase inhibitor (serpin), which specifically inhibits human tissue kallikrein by forming a covalent complex. The inhibitory activity of kallistatin is blocked upon its binding to heparin. In this study we attempted to locate the heparin-binding site of kallistatin using synthetic peptides derived from its surface regions and by site-directed mutagenesis of basic residues in these surface regions. Two synthetic peptides, containing clusters of positive-charged residues, one derived from the F helix and the other from the region encompassing the H helix and C2 sheet of kallistatin, were used to assess their heparin binding activity. Competition assay analysis showed that the peptide derived from the H helix and C2 sheet displayed higher and specific heparin binding activity. The basic residues in both regions were substituted to generate three kallistatin double mutants K187A/K188A (mutations in the F helix) and K307A/R308A and K312A/K313A (mutations in the region between the H helix and C2 sheet), using a kallistatin P1Arg variant as a scaffold. Analysis of these mutants by heparin-affinity chromatography showed that the heparin binding capacity of the variant K187A/K188A was not altered, whereas the binding capacity of K307A/R308A and K312A/K313A mutants was markedly reduced. Titration analysis with heparin showed that the K312A/K313A mutant has the highest dissociation constant. Like kallistatin, the binding activity of K187A/K188A to tissue kallikrein was blocked by heparin, whereas K307A/R308A and K312A/K313A retained significant binding and inhibitory activities in the presence of heparin. These results indicate that the basic residues, particularly Lys(312)-Lys(313), in the region between the H helix and C2 sheet of kallistatin, comprise a major heparin-binding site responsible for its heparin-suppressed tissue kallikrein binding.
authors.affiliation: Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, South Carolina 29425, USA.
authors.forename: V C
authors.initials: VC
authors.lastname: Chen
authors.forename: L
authors.initials: L
authors.lastname: Chao
authors.forename: D C
authors.initials: DC
authors.lastname: Pimenta
authors.forename: G
authors.initials: G
authors.lastname: Bledsoe
authors.forename: L
authors.initials: L
authors.lastname: Juliano
authors.forename: J
authors.initials: J
authors.lastname: Chao
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry* / Q000378:metabolism*; D002942:Circular Dichroism; D006493:Heparin / Q000378:metabolism*; D006801:Humans; D007610:Kallikreins / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D015843:Serpins / Q000737:chemistry* / Q000378:metabolism*; D012984:Software
chemical_list: D002352:Carrier Proteins; D015843:Serpins; C051324:kallistatin; D006493:Heparin; D007610:Kallikreins