Fluorescence Resonance Energy Transfer Study of Shape Changes in Membrane-Bound Bovine Prothrombin and Meizothrombin

Qing, Chen; Lentz, Barry R.

doi:10.1021/bi961441r

Cited by 29 publications

(28 citation statements)

References 35 publications

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“…It is therefore clear that the elongated APC protein binds to the membrane at one end and extends radially from the surface, a topographical motif that minimizes the phospholipid surface area occupied by the protein. Similar distances and conclusions have been obtained in previous studies with fVIIa (24), fIXa, (23), fXa (22,23), meizothrombin (25,26), and prothrombin (26). Thus, many of the proteolytic cleavages that effect and regulate hemostasis occur far above the membrane surface.…”

Section: Discussionsupporting

confidence: 87%

“…Our subsequent fluorescence energy transfer studies revealed that the active sites of factor Xa (fXa) (22,23), factor IXa (fIXa) (23), factor VIIa (fVIIa) (24), and meizothrombin (25), a prothombin derivative, are each located high above the membrane surface, thereby showing that the protease domain was located furthest from the bilayer in each case and confirming that the bound protein was oriented approximately perpendicularly to the plane of the membrane. The same conclusion was reached in another FRET study which showed that the protease domains of prothrombin and meizothrombin are both far above the surface, although in somewhat different locations (26). These spectroscopic results, obtained with membrane-bound proteins under near-physiological conditions, are completely compatible with the recently reported solid-state crystal structures of fIXa (27) and of fVIIa bound to a proteolyzed derivative of tissue factor (28).…”

supporting

confidence: 84%

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Protein S Alters the Active Site Location of Activated Protein C above the Membrane Surface

Yegneswaran¹,

Wood²,

Esmon³

et al. 1997

Journal of Biological Chemistry

101

140

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The location of the active site of membrane-bound activated protein C (APC) relative to the phospholipid surface was determined both in the presence and absence of its cofactor, protein S, using fluorescence resonance energy transfer (FRET). APC was chemically modified to create the FRET donor species, Fl-FPR-APC, with a fluorescein dye (Fl) covalently attached to the active site via a D-Phe-Pro-Arg (FPR) tether and located in the active site near S 4 . FRET was observed when Fl-FPR-APC was titrated in the presence of Ca 2؉ ions with phosphatidylcholine/phosphatidylserine (4:1) vesicles containing the FRET acceptor, octadecylrhodamine (OR). Assuming a random orientation of transition dipoles ( 2 ‫؍‬ 2/3), the average distance of closest approach between the fluorescein in the active site of the membrane-bound APC and the OR at the membrane surface is 94 Å. The same calcium-dependent distance was obtained for both small and large unilamellar vesicles and for vesicles that contained phosphatidylethanolamine. The active site of membrane-bound APC is therefore located far above the phospholipid surface. Upon addition of protein S, the efficiency of Fl-FPR-APC to OR energy transfer increased due to a protein S-dependent rotational and/or translational movement of the APC protease domain relative to the surface. If this movement were solely translational, then the average height of the fluorescein in the membrane-bound APC⅐protein S complex would be 84 Å above the surface. The extent of Fl-FPR-APC to OR energy transfer was unaltered by the addition of thrombin-inactivated protein S. The protein S effect was also specific for APC, since the addition of protein S to similarly-labeled derivatives of factor Xa, factor IXa, or factor VIIa did not alter the locations of their active sites. This direct measurement demonstrates that the binding of the protein S cofactor to its cognate enzyme elicits a relocation of the active site of APC relative to the membrane surface and thereby provides a structural explanation for the recently observed protein S-dependent change in the site of factor Va cleavage by APC.

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Section: Discussionsupporting

confidence: 87%

supporting

confidence: 84%

Protein S Alters the Active Site Location of Activated Protein C above the Membrane Surface

Yegneswaran¹,

Wood²,

Esmon³

et al. 1997

Journal of Biological Chemistry

101

140

View full text Add to dashboard Cite

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“…Additionally, cleavage of II TM to yield an mIIa derivative resistant to further proteolytic processing or the addition of prothrombinase to cleavage resistant II QQ was accompanied by relatively minor changes in light scattering intensity (Fig. 1B), consistent with the equivalent size of prothrombin and mIIa as well as the established ability of these species to bind PCPS with similar affinity (36,37). Our findings indicate that thrombin formation, irrespective of further processing at Arg 155 , leads to a decrease in inferred size of the PCPS vesicle.…”

Section: Changes In Vesicle Size Associated With Prothrombinmentioning

confidence: 65%

Fate of Membrane-bound Reactants and Products during the Activation of Human Prothrombin by Prothrombinase

Kamath

Krishnaswamy

2008

Journal of Biological Chemistry

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Membrane binding by prothrombin, mediated by its N-terminal fragment 1 (F1) domain, plays an essential role in its proteolytic activation by prothrombinase. Thrombin is produced in two cleavage reactions. One at Arg 320 yields the proteinase meizothrombin that retains membrane binding properties. The second, at Arg 271 , yields thrombin and severs covalent linkage with the N-terminal fragment 1.2 (F12) region. Covalent linkage with the membrane binding domain is also lost when prethrombin 2 (P2) and F12 are produced following initial cleavage at Arg 271 . We show that at the physiological concentration of prothrombin, thrombin formation results in rapid release of the proteinase into solution. Product release from the surface can be explained by the weak interaction between the proteinase and F12 domains. In contrast, the zymogen intermediate P2, formed following cleavage at Arg 271 , accumulates on the surface because of a ϳ20-fold higher affinity for F12. By kinetic studies, we show that this enhanced binding adequately explains the ability of unexpectedly low concentrations of F12 to greatly enhance the conversion of P2 to thrombin. Thus, the utilization of all three possible substrate species by prothrombinase is regulated by their ability to bind membranes regardless of whether covalent linkage to the F12 region is maintained. The product, thrombin, interacts with sufficiently poor affinity with F12 so that it is rapidly released from its site of production to participate in its numerous hemostatic functions.Thrombin, the key effector serine proteinase of the blood coagulation cascade, is produced by specific and limited proteolysis of the zymogen, prothrombin. The physiologically relevant catalyst for this reaction is the prothrombinase complex consisting of the serine proteinase, factor Xa, and the cofactor, factor Va, assembled on membranes in the presence of Ca 2ϩ(1). In addition to facilitating the assembly of the enzyme complex, membranes containing acidic or amino phospholipids play an important role in mediating the delivery of prothrombin to the membrane-bound enzyme (1, 2). This arises from the ability of prothrombin to bind to these membranes through the fragment 1 (F1) 2 domain present at its N terminus (1, 3, 4). Thrombin, derived from the C-terminal half of prothrombin, is produced as a result of cleavages 3 following Arg 271 and Arg 320(1, 3, 5). Cleavage at Arg 320 converts the zymogen to a proteinase, whereas cleavage at Arg 271 severs covalent linkage with the N-terminal fragment 1.2 (F12) domain harboring the membrane binding site (Scheme 1). Covalent linkage of the C-terminal domain with F12 is also lost in the zymogen intermediate, prethrombin 2 (P2), produced following cleavage only at Arg 271 (Scheme 1). In contrast, meizothrombin (mIIa), produced following cleavage only at Arg 320 is covalently linked to the membrane binding domain through a disulfide bond (Scheme 1). Accordingly, both prothrombin and mIIa are established to bind to membranes, and this binding interaction impacts the...

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“…These changes are associated with the formation of the substrate binding pocket and the oxyanion hole required for catalysis (11)(12)(13)(14)22). In the case of mIIa, these conformational changes could also be transmitted to the other domains that remain covalently attached (25). Because **Residues in the proteinase domain, numbered according to the homologous residues in chymotrypsinogen (22), are denoted by a C preceding the residue number.…”

Section: Discussionmentioning

confidence: 99%

Ratcheting of the substrate from the zymogen to proteinase conformations directs the sequential cleavage of prothrombin by prothrombinase

Bianchini

Orcutt

Panizzi

et al. 2005

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

119

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Prothrombinase catalyzes thrombin formation by the ordered cleavage of two peptide bonds in prothrombin. Although these bonds are likely Ϸ36 Å apart, sequential cleavage of prothrombin at Arg-320 to produce meizothrombin, followed by its cleavage at Arg-271, are both accomplished by equivalent exosite interactions that tether each substrate to the enzyme and facilitate presentation of the scissile bond to the active site of the catalyst. We show that impairing the conformational transition from zymogen to active proteinase that accompanies the formation of meizothrombin has no effect on initial cleavage at Arg-320 but inhibits subsequent cleavage at Arg-271. Full thermodynamic rescue of this defective mutant was achieved by stabilizing the proteinase-like conformation of the intermediate with a reversible, active sitespecific inhibitor. Irreversible stabilization of intact prothrombin in a proteinase-like state, even without prior cleavage at Arg-320, also enhanced cleavage at Arg-271. Our results indicate that the sequential presentation and cleavage of the two scissile bonds in prothrombin activation is accomplished by substrate bound either in the zymogen or proteinase conformations. The ordered cleavage of prothrombin by prothrombinase is driven by ratcheting of the substrate from the zymogen to the proteinase-like states.blood coagulation ͉ enzymology ͉ proteolytic cleavage ͉ serine protease ͉ zymogen activation T hrombin (IIa) is produced in a pivotal reaction of the blood coagulation cascade by specific proteolysis of prothrombin at two sites (1). The membrane-assembled prothrombinase complex is considered the physiologically relevant catalyst for this reaction (1, 2). Prothrombinase acts on the two scissile bonds through sequential cleavage reactions in an apparently ordered fashion. Provided that membrane binding by prothrombin is not compromised, prothrombin is cleaved after Arg-320 to yield meizothrombin ¶ (mIIa) as an intermediate, followed by subsequent cleavage at Arg-271 to produce IIa (3, 4). The molecular bases for these findings remain obscure.Because both cleavage sites in the zymogen appear accessible to proteolysis, prothrombin can be converted to thrombin by two possible cleavage pathways (3-6). Substrates for each of the four possible half-reactions in prothrombin activation bind prothrombinase in a mutually exclusive fashion and with equivalent affinity (4, 7). Three of the four possible reactions exhibit identical catalytic efficiencies, whereas cleavage at Arg-271 in intact prothrombin proceeds with a V max that is Ϸ30-fold lower (4). These findings establish the quantitative basis for preferential cleavage at Arg-320 over Arg-271 in intact prothrombin and the largely ordered cleavage pathway that ensues (4). They fail, however, to provide an explanation for why a difference in V max , and not affinity, drives selective recognition of the Arg-320 bond in intact prothrombin or why initial cleavage at Arg-320 greatly enhances the V max for subsequent cleavage at Arg-271 without also affec...

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Fluorescence Resonance Energy Transfer Study of Shape Changes in Membrane-Bound Bovine Prothrombin and Meizothrombin

Cited by 29 publications

References 35 publications

Protein S Alters the Active Site Location of Activated Protein C above the Membrane Surface

Protein S Alters the Active Site Location of Activated Protein C above the Membrane Surface

Fate of Membrane-bound Reactants and Products during the Activation of Human Prothrombin by Prothrombinase

Ratcheting of the substrate from the zymogen to proteinase conformations directs the sequential cleavage of prothrombin by prothrombinase

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