Localization of the Heparin Binding Exosite of Factor IXa

Yang, Likui; Manithody, Chandrashekhara; Rezaie, Alireza R.

doi:10.1074/jbc.m208485200

Cited by 61 publications

(68 citation statements)

References 31 publications

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“…Intriguingly, the location of the Fab75 epitope closely corresponds to thrombin exosite II, a positively charged region that interacts with allosteric effector molecules, such as heparin and thrombomodulin (24). Thrombin exosite II and the related heparin-binding exosites on coagulation factors IX (25) and X (26) comprise a cluster of functionally important arginine and lysine residues (25)(26)(27)(28)(29), which is not the case for the Ab75 epitope.…”

Section: Discussionmentioning

confidence: 99%

Structural insight into distinct mechanisms of protease inhibition by antibodies

Wu¹,

Eigenbrot

Liang³

et al. 2007

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

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To better understand how the relatively flat antigen-combining sites of antibodies interact with the concave shaped substrate-binding clefts of proteases, we determined the structures of two antibodies in complex with the trypsin-like hepatocyte growth-factor activator (HGFA). The two inhibitory antibodies, Ab58 and Ab75, were generated from a human Fab phage display library with synthetic diversity in the three complementarity determining regions (H1, H2, and H3) of the heavy chain, mimicking the natural diversity of the human Ig repertoire. Biochemical studies and the structures of the Fab58:HGFA (3.5-Å resolution) and the Fab75:HGFA (2.2-Å resolution) complexes revealed that Ab58 obstructed substrate access to the active site, whereas Ab75 allosterically inhibited substrate hydrolysis. In both cases, the antibodies interacted with the same protruding element (99-loop), which forms part of the substrate-binding cleft. Ab58 inserted its H1 and H2 loops in the cleft to occupy important substrate interaction sites (S3 and S2). In contrast, Ab75 bound at the backside of the cleft to a region corresponding to thrombin exosite II, which is known to interact with allosteric effector molecules. In agreement with the structural analysis, binding assays with active site inhibitors and enzymatic assays showed that Ab58 is a competitive inhibitor, and Ab75 is a partial competitive inhibitor. These results provide structural insight into antibody-mediated protease inhibition. They suggest that unlike canonical inhibitors, antibodies may preferentially target protruding loops at the rim of the substrate-binding cleft to interfere with the catalytic machinery of proteases without requiring long insertion loops.catalysis ͉ enzyme ͉ phage display

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Section: Discussionmentioning

confidence: 99%

Structural insight into distinct mechanisms of protease inhibition by antibodies

Wu¹,

Eigenbrot

Liang³

et al. 2007

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

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“…We also observed a surprising heparin interaction in the form of a crystallographic contact between the pentasaccharide and fIXa. The heparin-binding site of fIXa has been inferred from sequence homology to other heparin-binding proteases (exosite II), such as thrombin and fXa, and the contribution of several basic residues was determined by mutagenesis (12). We find that the pentasaccharide binds in the known heparin-binding site of fIXa, utilizing some of the previously implicated residues (Fig.…”

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confidence: 97%

“…This conformational change has been described in detail elsewhere (10,11), but functionally it amounts to the expulsion of the N-terminal portion (hinge region) of the reactive center loop (RCL) from β-sheet A to effectively increase its length. Factors IXa and Xa have similar heparin-binding sites to the well-characterized exosite II of thrombin (12,13), and, therefore, long heparin chains can provide additional acceleration through the template effect. Crystal structures of the heparin-activated AT Michaelis complexes with thrombin and fXa have been solved (14,15), illustrating why thrombin inhibition is not allosterically activated and why fXa recognition requires the pentasaccharide-induced conformational change in AT.…”

mentioning

confidence: 99%

Molecular basis of factor IXa recognition by heparin-activated antithrombin revealed by a 1.7-Å structure of the ternary complex

Johnson

Langdown

Huntington

2009

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

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Factor (f) IXa is a critical enzyme for the formation of stable blood clots, and its deficiency results in hemophilia. The enzyme functions at the confluence of the intrinsic and extrinsic pathways by binding to fVIIIa and rapidly generating fXa. In spite of its importance, little is known about how fIXa recognizes its cofactor, its substrate, or its only known inhibitor, antithrombin (AT). However, it is clear that fIXa requires extensive exosite interactions to present substrates for efficient cleavage. Here we describe the 1.7-Å crystal structure of fIXa in its recognition (Michaelis) complex with heparin-activated AT. It represents the highest resolution structure of both proteins and allows us to address several outstanding issues. The structure reveals why the heparin-induced conformational change in AT is required to permit simultaneous active-site and exosite interactions with fIXa and the nature of these interactions. The reactive center loop of AT has evolved to specifically inhibit fIXa, with a P2 Gly so as not to clash with Tyr99 on fIXa, a P4 Ile to fit snugly into the S4 pocket, and a C-terminal extension to exploit a unique wall-like feature of the active-site cleft. Arg150 is at the center of the exosite interface, interacting with AT residues on β-sheet C. A surprising crystal contact is observed between the heparin pentasaccharide and fIXa, revealing a plausible mode of binding that would allow longer heparin chains to bridge the complex.hemophilia | hemostasis | pentasaccharide | protease | thrombosis B lood coagulation (hemostasis) is traditionally described as two separate cascades of proteolytic activation events, the so-called intrinsic and extrinsic pathways (1, 2) (for a recent review, see ref.3). The extrinsic pathway is initiated by tissue damage that exposes tissue factor (TF) and subendothelial matrix proteins to the blood. Platelets adhere to collagen, and circulating factor (f) VIIa binds to and is activated by TF. The fVIIa-TF complex (extrinsic Xase) activates fX, and fXa in the presence of fVa produces thrombin. Just enough thrombin is generated at this stage for the formation of an initial clot through platelet activation; however, unless much more thrombin is produced in short order, the clot will not persist and bleeding will result. The second stage of hemostasis utilizes components of the intrinsic pathway, factors VIIIa and IXa, and deficiency of these critical proteins is the cause of hemophilia A and B, respectively. The fIXa-fVIIIa complex is known as intrinsic Xase and forms on the surface of activated platelets to efficiently produce fXa, resulting in a burst of thrombin formation.Regulatory proteins guard against overgrowth or dissemination of the clot, and all proteases generated in the cascade must eventually be inhibited. Factors VIIa and Xa are inhibited during the initiation phase by tissue factor pathway inhibitor (4), a triple Kunitz-domain canonical inhibitor, but the principal inhibitor of the coagulation proteases is the aptly named member of the serpin fam...

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“…After 50 min of activation at an ambient temperature, the reactions were terminated by adding 50 mM EDTA. The rate of factor IXa generation was determined by an amidolytic activity assay using CBS 31.39 containing 33% ethylene glycol as described previously (17). The initial rates of chromogenic substrate hydrolysis (⌬A 405 /min) were converted to nanomolar of product by reference to a standard curve prepared with purified human factor IXa.…”

Section: Evaluation Of the K D(app)mentioning

confidence: 99%

The Cofactor Function of the N-terminal Domain of Tissue Factor

Kittur

Manithody

Morrissey

et al. 2004

Journal of Biological Chemistry

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Tissue factor (TF) is an integral membrane protein cofactor for factor VIIa (fVIIa) that initiates the blood coagulation cascade during vascular injury. TF has two fibrinonectin type III-like domains, both of which make extensive interactions with both the light and heavy chains of fVIIa. In addition to interaction with fVIIa, the membrane proximal C-terminal domain of TF is also known to bind the natural substrates factors IX and X, thereby facilitating their assembly and recognition by fVIIa in the activation complex. Both fVIIa and TF are elongated proteins, and their complex appears to be positioned nearly perpendicular to the membrane surface. It is possible that, similar to fVIIa, the N-terminal domain of TF also contacts the natural substrates. To investigate this possibility, we substituted all 23 basic and acidic residues of the N-terminal domain of TF with Ala or Asn and expressed the mutants as soluble TF 2-219 in a novel expression/purification vector system in the periplasmic space of bacteria. Following purification to homogeneity, the cofactor properties of mutants in promoting the amidolytic and proteolytic activity of fVIIa were analyzed in appropriate kinetic assays. The amidolytic activity assays indicated that several charged residues spatially clustered at the junction of the N-and C-terminal domains of TF are required for high affinity interaction with fVIIa. On the other hand, the proteolytic activity assays revealed that none of the residues under study may be an interactive site for either factor IX or factor X. However, it was discovered the Arg 74 mutant of TF was defective in enhancing both the amidolytic and proteolytic activity of fVIIa, suggesting that this residue may be required for the allosteric activation of the protease.Tissue factor (TF) 1 is an integral membrane cofactor that upon exposure to circulating blood binds with high affinity to factor VIIa (fVIIa) to catalyze the rapid activation of procoagulant zymogens factors IX and X, thereby initiating the blood clotting cascade (1-3). The structure of TF is composed of two fibrinonectin type III-like extracellular domains, a single membrane-spanning domain and a short cytoplasmic tail (4, 5). Although all three domains of TF are required for the physiological function of the cofactor, previous studies have indicated that the two extracellular domains expressed by recombinant DNA methods as a soluble protein (sTF) can bind to fVIIa with a high affinity to enhance both the amidolytic and proteolytic activities of the protease (6 -9). The crystal structure of sTF either alone or in complex with fVIIa has been determined (4, 10). The structural data have indicated that the extracellular domains of TF make extensive interactions with both the light and heavy chains of fVIIa (10). It is believed that these interactions allosterically change the conformation of the active-site pocket of fVIIa, leading to a dramatic improvement in the catalytic efficiency of the protease toward both synthetic and natural macromolecular substrates (1...

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Localization of the Heparin Binding Exosite of Factor IXa

Abstract: Factor IXa (FIXa) is known to have a binding site for heparin that has not been mapped by a mutagenesis study. By homology modeling based on structural data, we identified eight basic residues in the catalytic domain of FIXa that can potentially bind to heparin.

Cited by 61 publications

References 31 publications

Structural insight into distinct mechanisms of protease inhibition by antibodies

Structural insight into distinct mechanisms of protease inhibition by antibodies

Molecular basis of factor IXa recognition by heparin-activated antithrombin revealed by a 1.7-Å structure of the ternary complex

The Cofactor Function of the N-terminal Domain of Tissue Factor

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