The Location of the Active Site of Blood Coagulation Factor VIIa above the Membrane Surface and Its Reorientation upon Association with Tissue Factor

McCallum, Christine; Hapak, Raymond C.; Neuenschwander, Pierre F.; Morrissey, James H.; Johnson, Arthur E.

doi:10.1074/jbc.271.45.28168

Cited by 73 publications

(89 citation statements)

References 44 publications

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“…Although the position of the active site above the membrane surface has been postulated to be functionally important (12,13,15,16,43), the data reported here constitute the first direct demonstration that modulation of active site location can alter enzyme activity and that the cofactor can be rendered irrelevant by the appropriate repositioning of the active site. Thus, we conclude that protein-dependent modulation of topography (specifically, structure relative to the membrane surface) is an effective means to regulate the activity of membrane-bound enzymes involved in hemostasis and likely in other membranedependent processes.…”

Section: Discussionmentioning

confidence: 88%

“…For example, tissue factor was found to alter the location of the active site of membrane-bound factor VIIa relative to the membrane surface (15), and this position was dictated by the cofactor, not by the membrane binding Gla domain of factor VIIa (39). Also, factor Va binding to factor Xa on a membrane surface causes translocation and/or rotation of the active site of the enzyme relative to the membrane surface (13).…”

Section: Discussionmentioning

confidence: 99%

“…Light scattering experiments indicated that two elongated vitamin K-dependent proteins, prothrombin (PT) and factor X, project radially from the surface when bound to the membrane (11), and our fluorescence resonance energy transfer (FRET) experiments showed that the active site of each of the vitamin K-dependent enzymes is located far (Ͼ70 Å) above the membrane surface (12)(13)(14)(15)(16), thereby indicating that they project approximately perpendicularly from the membrane surface. In the case of membrane-bound APC, its active site is located an average of 94 Å above the surface (assuming 2 ϭ 2/3; Ref.…”

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confidence: 98%

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Relocating the Active Site of Activated Protein C Eliminates the Need for Its Protein S Cofactor

Yegneswaran¹,

Smirnov²,

Safa³

et al. 1999

Journal of Biological Chemistry

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The effect of replacing the ␥-carboxyglutamic acid domain of activated protein C (APC) with that of prothrombin on the topography of the membrane-bound enzyme was examined using fluorescence resonance energy transfer. The average distance of closest approach (assuming 2 ‫؍‬ 2/3) between a fluorescein in the active site of the chimera and octadecylrhodamine at the membrane surface was 89 Å, compared with 94 Å for wildtype APC. The ␥-carboxyglutamic acid domain substitution therefore lowered and/or reoriented the active site, repositioning it close to the 84 Å observed for the APC⅐protein S complex. Protein S enhances wild-type APC cleavage of factor Va at Arg 306 , but the inactivation rate of factor Va Leiden by the chimera alone is essentially equal to that by wild-type APC plus protein S. These data suggest that the activities of the chimera and of the APC⅐protein S complex are equivalent because the active site of the chimeric protein is already positioned near the optimal location above the membrane surface to cleave Arg 306 . Thus, one mechanism by which protein S regulates APC activity is by relocating its active site to the proper position above the membrane surface to optimize factor Va cleavage.

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Section: Discussionmentioning

confidence: 88%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 98%

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Relocating the Active Site of Activated Protein C Eliminates the Need for Its Protein S Cofactor

Yegneswaran¹,

Smirnov²,

Safa³

et al. 1999

Journal of Biological Chemistry

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“…This conclusion is true for both point-to-point (e.g., refs. 17, 21, 25, and 26) and plane-toplane (18,23,27,28) FRET measurements. Furthermore, because Rh-PE is oriented randomly in the plane of the membrane in the plane-to-plane FRET measurements done here, the uncertainty in R 0 due to orientation effects is further reduced.…”

Section: Dye Orientation ( 2 ) Effects and Uncertainties In Lmentioning

confidence: 99%

The domains of a cholesterol-dependent cytolysin undergo a major FRET-detected rearrangement during pore formation

Ramachandran

Tweten

Johnson

2005

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

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FRET measurements were used to determine the domain-specific topography of perfringolysin O, a pore-forming toxin, on a membrane surface at different stages of pore formation. The data reveal that the elongated toxin monomer binds stably to the membrane in an ''end-on'' orientation, with its long axis approximately perpendicular to the plane of the membrane bilayer. This orientation is largely retained even after monomer association to form an oligomeric prepore complex. The domain 3 (D3) polypeptide segments that ultimately form transmembrane ␤-hairpins remain far above the membrane surface in both the membrane-bound monomer and prepore oligomer. Upon pore formation, these segments enter the bilayer, whereas D1 moves to a position that is substantially closer to the membrane. Therefore, the extended D2 ␤-structure that connects D1 to membrane-bound D4 appears to bend or otherwise reconfigure during the prepore-to-pore transition of the perfringolysin O oligomer. membrane protein ͉ toxin ͉ fluorescence ͉ membrane P erfringolysin O (PFO), a cytolytic toxin from the pathogenic bacterium Clostridium perfringens, perforates cholesterolcontaining eukaryotic cell membranes by forming large aqueous pores that measure up to 300 Å in diameter (1). PFO belongs to a large family of protein toxins termed the ''cholesteroldependent cytolysins'' (CDCs) that serve as potent virulence factors for various pathogenic Gram-positive bacteria (2). Pore formation is accomplished by a complex mechanism that includes the stable binding of water-soluble PFO monomers to membranes with sufficient cholesterol, lateral diffusion of monomers on the membrane surface, the association of monomers into oligomeric prepore complexes that are composed of up to 50 polypeptides, and then the concerted insertion of a large oligomeric amphipathic ␤-barrel as the prepore complex enters the membrane bilayer to create a large aqueous pore (3, 4).The transition of the water-soluble PFO monomer into a membrane-inserted oligomer involves extensive changes in protein conformation that have been the recent focal point of research. Several structural states and rearrangements have been identified, along with various protein-lipid and protein-protein interactions that mediate the conformational changes, by using the crystal structure of the PFO monomer (5), a repertoire of site-specifically mutagenized PFO derivatives, and multiple independent fluorescence techniques (6). Domain 4 (D4) is located at one end of the elongated PFO monomer (Fig. 1A) and is responsible for membrane recognition and initial binding (7), but only the tip of this domain is embedded in the nonpolar interior of the bilayer (8). D4-lipid interactions trigger conformational changes in the spatially distant D3 that expose a previously hidden interface for oligomerization and, hence, prepore complex formation (9). Two sets of three short ␣-helices in D3 then undergo an ␣-helix-to-␤-sheet transition to create two transmembrane ␤-hairpins (TMHs), TMH1 and TMH2, per monomer that are then inserted...

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“…It has been hypothesized that an important role for membrane-bound cofactors such as TF is to fix the active site of their cognate protease at the optimal position and orientation relative to the membrane surface for attack on the scissile bonds of membrane-bound protein substrates (10,11). Using fluorescence resonance energy transfer (RET) techniques, we previously showed that when fVIIa binds to phospholipid membranes, its active site is located 83 Å above the membrane surface, but this distance shortens to about 75 Å when fVIIa binds to TF (11). This was true even when the fVIIa Gla domain was removed, demonstrating that TF alone is sufficient to dictate the location of the fVIIa active site above the membrane surface (12).…”

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confidence: 99%

Raising the Active Site of Factor VIIa above the Membrane Surface Reduces Its Procoagulant Activity but Not Factor VII Autoactivation

Waters

Yegneswaran

Morrissey

2006

Journal of Biological Chemistry

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Tissue factor, the physiologic trigger of blood clotting, is the membrane-anchored protein cofactor for the plasma serine protease, factor VIIa. Tissue factor is hypothesized to position and align the active site of factor VIIa relative to the membrane surface for optimum proteolytic attack on the scissile bonds of membrane-bound protein substrates such as factor X. We tested this hypothesis by raising the factor VIIa binding site above the membrane surface by creating chimeras containing the tissue factor ectodomain linked to varying portions of the membraneanchored protein, P-selectin. The tissue factor/P-selectin chimeras bound factor VIIa with high affinity and supported full allosteric activation of factor VIIa toward tripeptidyl-amide substrates. That the active site of factor VIIa was raised above the membrane surface when bound to tissue factor/P-selectin chimeras was confirmed using resonance energy transfer techniques in which appropriate fluorescent dyes were placed in the active site of factor VIIa and at the membrane surface. The chimeras were deficient in supporting factor X activation by factor VIIa due to decreased k cat . The chimeras were also markedly deficient in clotting plasma, although incubating factor VII or VIIa with the chimeras prior to the addition of plasma restored much of their procoagulant activity. Interestingly, all chimeras fully supported tissue factor-dependent factor VII autoactivation. These studies indicate that proper positioning of the factor VII/VIIa binding site on tissue factor above the membrane surface is important for efficient rates of activation of factor X by this membrane-bound enzyme/cofactor complex. Tissue factor (TF)3 is the cell surface, type I integral membrane protein that triggers blood clotting in normal hemostasis and many thrombotic diseases (1, 2). TF initiates blood coagulation by binding the plasma serine protease, factor VIIa (fVIIa). The membrane-bound complex of fVIIa and TF can therefore be considered a two-subunit enzyme, with fVIIa as the catalytic subunit and TF as the regulatory subunit. The major substrates for the fVIIa-TF complex are factors X (fX) and IX (fIX), which are converted to active serine proteases by limited proteolysis. Activation of fX and fIX leads to propagation of the clotting cascade and, ultimately, clotted fibrin and activated platelets. Binding of fVIIa to TF embedded in a suitable phospholipid membrane increases its proteolytic activity many thousandfold (3, 4). For optimal fVIIa-TF enzymatic activity, the phospholipid bilayer must contain negatively charged phospholipid, most especially phosphatidylserine (PS) (5, 6). How PS augments the enzymatic activity of fVIIa-TF is not completely understood, but one role is to promote interaction of vitamin K-dependent clotting factors such as fVII, fIX, and fX with phospholipid surfaces (7). These proteins all have an N-terminal domain containing multiple ␥-carboxyglutamate residues (Gla domain) that confers reversible binding to membranes containing PS. Incorporating PS in...

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The Location of the Active Site of Blood Coagulation Factor VIIa above the Membrane Surface and Its Reorientation upon Association with Tissue Factor

Cited by 73 publications

References 44 publications

Relocating the Active Site of Activated Protein C Eliminates the Need for Its Protein S Cofactor

Relocating the Active Site of Activated Protein C Eliminates the Need for Its Protein S Cofactor

The domains of a cholesterol-dependent cytolysin undergo a major FRET-detected rearrangement during pore formation

Raising the Active Site of Factor VIIa above the Membrane Surface Reduces Its Procoagulant Activity but Not Factor VII Autoactivation

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