Importance of Lysine 125 for Heparin Binding and Activation of Antithrombin

Schedin‐Weiss, Sophia; Desai, Umesh R.; Bock, Susan; Gettins, Peter G.W.; Olson, Steven T.; Björk, Ingemar

doi:10.1021/bi012163l

Cited by 47 publications

(76 citation statements)

References 42 publications

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“…1d compares the heparin binding sites of AT and HCII, with key residues indicated. The critical residues involved in the tight binding of the heparin pentasaccharide by AT have been demonstrated by both structural and biochemical studies to include Lys-114, Lys-125, and Arg-129, whereas Arg-132, Lys-133, and Lys-136 contribute to the binding of longer heparin chains (8,(29)(30)(31)(32)(33). The conservation in HCII of the key residues involved in the interaction of the pentasaccharide with AT supports a similar mechanism of GAG binding by HCII.…”

Section: Resultsmentioning

confidence: 74%

Crystal structures of native and thrombin-complexed heparin cofactor II reveal a multistep allosteric mechanism

Baglin

Carrell

Church

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

188

196

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The serine proteases sequentially activated to form a fibrin clot are inhibited primarily by members of the serpin family, which use a unique ␤-sheet expansion mechanism to trap and destroy their targets. Since the discovery that serpins were a family of serine protease inhibitors there has been controversy as to the role of conformational change in their mechanism. It now is clear that protease inhibition depends entirely on rapid serpin ␤-sheet expansion after proteolytic attack. The regulatory advantage afforded by the conformational mobility of serpins is demonstrated here by the structures of native and S195A thrombin-complexed heparin cofactor II (HCII). HCII inhibits thrombin, the final protease of the coagulation cascade, in a glycosaminoglycan-dependent manner that involves the release of a sequestered hirudin-like N-terminal tail for interaction with thrombin. The native structure of HCII resembles that of native antithrombin and suggests an alternative mechanism of allosteric activation, whereas the structure of the S195A thrombin-HCII complex defines the molecular basis of allostery. Together, these structures reveal a multistep allosteric mechanism that relies on sequential contraction and expansion of the central ␤-sheet of HCII.T he predominant protease inhibitors of the higher organisms, the serpins (1, 2), have evolved a complex mechanism that was revealed recently by the crystallographic structure of a serpin-protease complex (3). This dramatic mechanism amounts to a race between proteolysis and the incorporation of the serpin-reactive center loop into its main ␤-sheet (␤-sheet A). The dependence of the serpin inhibitory mechanism on ␤-sheet expansion renders serpins highly susceptible to both loss-offunction and gain-of-function diseases but additionally allows for a range of mechanisms for the modulation of serpin activity. Thus, serpins typically are found controlling tightly regulated physiological pathways such as blood coagulation. Antithrombin (AT) and heparin cofactor II (HCII) have independently (4, 5) evolved mechanisms by which they circulate in an inert state until activated by binding to cell surface glycosaminoglycans (GAGs). AT and HCII thus are allosterically activated toward factor Xa and thrombin, respectively, the final two proteases in the blood coagulation cascade.The structure and mechanism of activation of AT has been well characterized. Its fold is similar to that of the rest of the serpin family with the single and important exception that the reactive center loop is constrained through partial incorporation into ␤-sheet A (refs. 6 and 7; Fig. 1a). The native conformation of AT thus renders it incapable of forming a productive recognition complex (Michaelis complex) with factor Xa, and only as the result the conformational changes brought about through the interaction with a specific heparin pentasaccharide sequence does AT become an efficient inhibitor of factor Xa. Pentasaccharide binding results in secondary structural changes in the heparin binding region and the ex...

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Section: Resultsmentioning

confidence: 74%

Crystal structures of native and thrombin-complexed heparin cofactor II reveal a multistep allosteric mechanism

Baglin

Carrell

Church

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

188

196

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“…24 The corresponding glycoform of K125M antithrombin lacking carbohydrate at N135 was isolated. 21 Both low-and high-heparin affinity glycoforms of wild-type and K114M recombinant antithrombins that were or were not fucosylated at the Asn 155 glycosylation site were isolated by heparin-agarose chromatography. [25][26][27] The cleaved form of recombinant antithrombins was prepared by treatment with neutrophil elastase followed by successive heparinSepharose and MonoQ chromatography steps.…”

Section: Antithrombin and Heparinsmentioning

confidence: 99%

“…[24][25][26][27] Mutations of Lys125 or Lys114 to Met result in substantial 200-fold and 10 5 -fold reductions in the affinity of native antithrombin for heparin, respectively. 21,22 These mutations reduced the affinity of cleaved antithrombin for heparin as well, as judged from the decreased salt concentrations at which the cleaved mutants eluted from heparin-agarose relative to the cleaved wild-type serpin (Table 1). However, the Lys125 and Lys114 mutations caused comparable losses in the affinity of cleaved antithrombin for heparin, in sharp contrast to the very different heparin affinity losses produced by these mutations in native antithrombin (Table 1).…”

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confidence: 99%

The heparin-binding site of antithrombin is crucial for antiangiogenic activity

Zhang

Swanson

Izaguirre

et al. 2005

Blood

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The heparin-binding site of antithrombin is shown here to play a crucial role in mediating the antiangiogenic activity of conformationally altered cleaved and latent forms of the serpin. Blocking the heparin-binding site of cleaved or latent antithrombin by complexation with a highaffinity heparin pentasaccharide abolished the serpin's ability to inhibit proliferation, migration, capillary-like tube formation, basic fibroblast growth factor (bFGF) signaling, and perlecan gene expression in bFGF-stimulated human umbilical vein endothelial cells. Mutation of key heparin binding residues, when combined with modifications of Asn-linked carbohydrate chains near the heparinbinding site, also could abrogate the antiproliferative activity of the cleaved serpin. Surprisingly, mutation of Lys114, which blocks anticoagulant activation of antithrombin by heparin, caused the native protein to acquire antiproliferative activity without the need for conformational change. Together, these results indicate that the heparin-binding site of antithrombin is of crucial importance for mediating the serpin's antiangiogenic activity and that heparin activation of native antithrombin constitutes an antiangiogenic switch that is responsible for turning off the antiangiogenic activity of the native serpin. IntroductionAntithrombin is an abundantly expressed, key plasma protein regulator of blood coagulation in vertebrates. 1 Inherited or acquired deficiencies of the protein in humans are associated with an increased risk of developing thrombotic disease, 2 and complete deficiency in mice results in embryonic lethality due to a consumptive coagulopathy. 3 Antithrombin regulates blood coagulation by directly inhibiting the serine proteases of the clotting cascade, the most important targets being thrombin, factor Xa, and factor IXa. 1,4 The protein is a member of the serpin superfamily of protein protease inhibitors and shares a common tertiary structure with the family. 5 It is unusual among serpins in requiring activation by the sulfated glycosaminoglycans, heparin or heparan sulfate, to inhibit its target proteases at a physiologically significant rate. A sequencespecific pentasaccharide present in only a fraction of heparin molecules mediates high-affinity binding and anticoagulant activation of antithrombin by the polysaccharide. 1,[6][7][8] In addition to its well-established anticoagulant function, antithrombin has more recently been shown to have anti-inflammatory, 9 antiviral, 10 and antiangiogenic functions. 11,12 The antiangiogenic activity has been demonstrated from the ability to inhibit basic fibroblast growth factor (bFGF)-or vascular endothelial growth factor (VEGF)-induced endothelial cell proliferation, blood vessel growth in the chick embryo, and tumor growth in mice. The antitumor activity equals or exceeds that of other well-established antiangiogenic agents. 13 Of importance, antiangiogenic activity is not present in native antithrombin but is expressed only after the protein undergoes conformational alterations induc...

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“…ATIII helix D (1) contains heparin-binding residues Lys 125 (5), Arg 129 (6), Phe 122 (7), Arg 132 , Lys 133 (8), and Lys 136 (9). Helix D of pentasaccharide-bound, RCL-expelled and activated ATIII (AT*H) (Fig.…”

mentioning

confidence: 99%

Disruption of a Tight Cluster Surrounding Tyrosine 131 in the Native Conformation of Antithrombin III Activates It for Factor Xa Inhibition

Cruz¹,

Jairajpuri²,

Bock³

2006

J. Biol. Chem.

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The native conformation of antithrombin III (ATIII) is a poor inhibitor of its coagulation pathway target enzymes because of the partial insertion of its reactive center loop (RCL) in its central A ␤-sheet. This study focused on tyrosine 131, which is located at the helix D-sheet A interface, adjacent to the ATIII pentasaccharide and heparin cofactor-binding sites and some 17 Å away from the RCL insertion. cluster at the helix D-strand 2A interface of native antithrombin contributes significantly to the stability of the ground state conformation, and tyrosine 131 serves as a heparin-responsive molecular switch during the allosteric activation of ATIII anticoagulant activity.To efficiently inhibit factor Xa (fXa), 2 the anticoagulant protein antithrombin III (ATIII) must first bind a pentasaccharide sequence found in pharmaceutical heparins or vascular wall heparan sulfate proteoglycans (HSPGs) and then undergo a protein conformational change that increases reactivity of its reactive center loop (RCL) and other recognition elements with the target enzyme (1-4). Protein conformational changes like the one underlying heparin-dependent ATIII activation result from reductions in the strengths of an initial set of native ground state structural interactions, which are coupled to increases in the strengths of new structural interactions formed during the course of activation. There may be an uncatalyzed equilibrium between different conformations of a protein. For example, the equilibrium between the RCL-inserted and RCLexpelled forms of ATIII is responsible for its slow progressive inhibition of clotting factors and is an example of an uncatalyzed equilibrium. Alternatively, binding of a cofactor ligand may drive the conformational equilibrium of a protein toward a particular state of the molecule, such as AT*H, the heparinbound and activated form of ATIII. For protein conformational changes driven by ligand binding, initial interactions between the ligand and the protein induce structural rearrangements proximal to the binding site, and these changes promote subsequent distal structural adjustments and conformational changes that are successively transmitted through the molecules until a stable complex is achieved.Our long term goal is to develop a thorough understanding of the allosteric, heparin binding-induced conformational change that propagates through ATIII and activates its anticoagulant activity. Intermediate steps in this project will be to identify key structural interactions that stabilize native, transient intermediate, and activated conformations of antithrombin and that are switched off or on during the activation process. This study describes the identification of one such switch, tyrosine 131, that is located at the interface of helix D and central ␤-sheet A.ATIII helix D (1) contains heparin-binding residues Lys 125 (5), Arg 129 (6), Phe 122 (7), Arg 132 , Lys 133 (8), and Lys 136 (9). Helix D of pentasaccharide-bound, RCL-expelled and activated ATIII (AT*H) (Fig. 1b) is 1.5 turns longer at its C-te...

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Importance of Lysine 125 for Heparin Binding and Activation of Antithrombin

Cited by 47 publications

References 42 publications

Crystal structures of native and thrombin-complexed heparin cofactor II reveal a multistep allosteric mechanism

Crystal structures of native and thrombin-complexed heparin cofactor II reveal a multistep allosteric mechanism

The heparin-binding site of antithrombin is crucial for antiangiogenic activity

Disruption of a Tight Cluster Surrounding Tyrosine 131 in the Native Conformation of Antithrombin III Activates It for Factor Xa Inhibition

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