Reformable intramolecular cross‐linking of the N‐terminal domain of heparin cofactor II

Abstract

The crystal structure of a heparin cofactor II (HCII)-thrombin Michaelis complex has revealed extensive contacts encompassing the N-terminal domain of HCII and exosite I of the proteinase. In contrast, the location of the N-terminal extension in the uncomplexed inhibitor was unclear. Using a disulfide cross-linking strategy, we demonstrate that at least three different sites (positions 52, 54 and 68) within the N terminus may be tethered in a reformable manner to position 195 in the loop region between helix D and strand s2A of the HCII molecule, suggesting that the N-terminal domain may interact with the inhibitor scaffold in a permissive manner. Cross-linking of the N terminus to the HCII body does not strongly affect the inhibition of a-chymotrypsin, indicating that the reactive site loop sequences of the engineered inhibitor variants, required for interaction with one of the HCII target enzymes, are normally accessible. In contrast, intramolecular tethering of the N-terminal extension results in a drastic decrease of a-thrombin inhibitory activity, both in the presence and in the absence of glycosaminoglycans. Treatment with dithiothreitol and iodoacetamide restores activity towards a-thrombin, suggesting that release of the N terminus of HCII is an important component of the multistep interaction between the inhibitor and a-thrombin.Keywords: a-thrombin; dermatan sulfate; heparin cofactor II; heparin; serpin(s).Heparin cofactor II (HCII), a member of the serpin family, is an efficient inhibitor of a-thrombin in the presence of a variety of polyanions, including glycosaminoglycan (GAGs) such as heparin or dermatan sulfate [1]. In the absence of these compounds, the rate of a-thrombin inhibition is lowered by several orders of magnitude. HCII also inhibits a-chymotrypsin [2] and cathepsin G [3]; the reaction rates, however, are only moderately affected by GAGs. In recent years, substantial evidence has been accumulated on the molecular basis underlying the inhibition of serine proteinases by serpins [4,5]. Key features of the mechanism include the presentation of the inhibitor's reactive site loop (RSL) to a target enzyme, the initial cleavage of the scissile bond within the RSL, and formation of a covalent acyl ester intermediate between the catalytic serine of the enzyme and the carboxyl group of the P1 residue of the RSL. In the inhibitory path of the branched pathway mechanism of serpins, the RSL is inserted into b-sheet A with concomitant translocation of the attached proteinase to the opposite pole of the inhibitor [6,7].For most serpins, the specificity of the inhibitor-enzyme reaction is primarily determined by residues within the RSL, with a dominant importance of residues flanking the scissile bond. However, exosite interactions can also play an important role, as recently demonstrated for the heparininduced acceleration of inhibition of factors Xa and IXa by antithrombin [8]. Exosite contacts have also been implicated in the GAG-enhanced a-thrombin inhibition by HCII [9][10][11], and the crystal st...

Heparin Cofactor II Modulates the Response to Vascular Injury

“…The rate of thrombin inhibition, particularly in the presence of a glycosaminoglycan, is greatly reduced by introduction of novel disulfide bonds that tether the N-terminal acidic domain to the body of HCII. 30 These findings are consistent with a model in which binding of heparin or DS to HCII induces a conformational …”

Functional diversification of a protease inhibitor gene in the genus Drosophila and its molecular basis

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Origin of Serpin-Mediated Regulation of Coagulation and Blood Pressure

“…Transfection of COS7 cells with polyethylenimine (PEI) was performed as outlined previously [21]. The lamprey angiotensinogen expression construct contained the human angiotensin II sequence in place of the original sequence thus enabling detection with anti-human angiotensin II antibodies [14].…”

“…Complex formation was assessed electrophoretically after heating (4 min at 95°C) under reducing conditions followed by immunoblotting [21]. Second-order rate constants of the FXa/Lfl_SpnV4_1 interaction were determined at 25°C by measuring cleavage of S-2222 at 405 nm in 50 mM Tris-HCl, 130 mM NaCl, 1 g/l PEG 8000, pH 8.3, in the presence or absence of heparin or Ca 2+ ions, using a 10-fold molar inhibitor excess as outlined previously [21]. Human FXa was titrated with antithrombin prior to determination of the reaction stoichiometry [24].…”