title: The complete N-terminal extension of heparin cofactor II is required for maximal effectiveness as a thrombin exosite 1 ligand.
abstract: BACKGROUND
  Heparin cofactor II (HCII) is a circulating protease inhibitor, one which contains an N-terminal acidic extension (HCII 1-75) unique within the serpin superfamily. Deletion of HCII 1-75 greatly reduces the ability of glycosaminoglycans (GAGs) to accelerate the inhibition of thrombin, and abrogates HCII binding to thrombin exosite 1. While a minor portion of HCII 1-75 can be visualized in a crystallized HCII-thrombin S195A complex, the role of the rest of the extension is not well understood and the affinity of the HCII 1-75 interaction has not been quantitatively characterized. To address these issues, we expressed HCII 1-75 as a small, N-terminally hexahistidine-tagged polypeptide in E. coli.
  
  
  RESULTS
  Immobilized purified HCII 1-75 bound active α-thrombin and active-site inhibited FPR-ck- or S195A-thrombin, but not exosite-1-disrupted γT-thrombin, in microtiter plate assays. Biotinylated HCII 1-75 immobilized on streptavidin chips bound α-thrombin and FPR-ck-thrombin with similar KD values of 330-340 nM. HCII 1-75 competed thrombin binding to chip-immobilized HCII 1-75 more effectively than HCII 54-75 but less effectively than the C-terminal dodecapeptide of hirudin (mean Ki values of 2.6, 8.5, and 0.29 μM, respectively). This superiority over HCII 54-75 was also demonstrated in plasma clotting assays and in competing the heparin-catalysed inhibition of thrombin by plasma-derived HCII; HCII 1-53 had no effect in either assay. Molecular modelling of HCII 1-75 correctly predicted those portions of the acidic extension that had been previously visualized in crystal structures, and suggested that an α-helix found between residues 26 and 36 stabilizes one found between residues 61-67. The latter region has been previously shown by deletion mutagenesis and crystallography to play a crucial role in the binding of HCII to thrombin exosite 1.
  
  
  CONCLUSIONS
  Assuming that the KD value for HCII 1-75 of 330-340 nM faithfully predicts that of this region in intact HCII, and that 1-75 binding to exosite 1 is GAG-dependent, our results support a model in which thrombin first binds to GAGs, followed by HCII addition to the ternary complex and release of HCII 1-75 for exosite 1 binding and serpin mechanism inhibition. They further suggest that, in isolated or transferred form, the entire HCII 1-75 region is required to ensure maximal binding of thrombin exosite 1.
authors.forename: Amanda J
authors.initials: AJ
authors.lastname: Boyle
authors.forename: Leigh Ann
authors.initials: LA
authors.lastname: Roddick
authors.forename: Varsha
authors.initials: V
authors.lastname: Bhakta
authors.forename: Melissa D
authors.initials: MD
authors.lastname: Lambourne
authors.forename: Murray S
authors.initials: MS
authors.lastname: Junop
authors.forename: Patricia C
authors.initials: PC
authors.lastname: Liaw
authors.forename: Jeffrey I
authors.initials: JI
authors.lastname: Weitz
authors.forename: William P
authors.initials: WP
authors.lastname: Sheffield
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites; D004926:Escherichia coli / Q000378:metabolism; D015844:Heparin Cofactor II / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006629:Hirudins / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism; D006639:Histidine / Q000235:genetics / Q000378:metabolism; D006801:Humans; D055767:Immobilized Proteins / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D051379:Mice; D056004:Molecular Dynamics Simulation; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000235:genetics / Q000378:metabolism; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D016415:Sequence Alignment; D015843:Serpins / Q000737:chemistry / Q000378:metabolism; D013917:Thrombin / Q000737:chemistry / Q000378:metabolism
chemical_list: D006629:Hirudins; C471213:His-His-His-His-His-His; D055767:Immobilized Proteins; D009842:Oligopeptides; D010455:Peptides; D011993:Recombinant Fusion Proteins; D015843:Serpins; D006639:Histidine; D015844:Heparin Cofactor II; D013917:Thrombin