Activation and Active Site Occupation Alter Conformation in the Region of the First Epidermal Growth Factor-like Domain of Human Factor VII

Leonard, Blair; Clarke, Bryan J.; Sridhara, Sampath; Kelley, Robert F.; Ofosu, Fred Kwame; Blajchman, Morris A.

doi:10.1074/jbc.m001166200

Cited by 19 publications

(21 citation statements)

References 44 publications

(46 reference statements)

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“…The functional consequences of this interchain transfer of an activation signal are at present unknown. Interestingly, the decreased HX rates in the EGF1 domain correlate with studies of an antibody specific for the EGF1 domain, which binds to FVIIa with increased affinity in the presence of a covalent active site inhibitor known to induce the active conformation (26). Furthermore, Ca 2ϩ -binding to the EGF1 domain has been shown to increase the amidolytic activity (27).…”

Section: Discussionmentioning

confidence: 83%

Allosteric Activation of Coagulation Factor VIIa Visualized by Hydrogen Exchange

Rand

Jørgensen

Olsen

et al. 2006

Journal of Biological Chemistry

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Coagulation factor VIIa (FVIIa) is a serine protease that, after binding to tissue factor (TF), plays a pivotal role in the initiation of blood coagulation. We used hydrogen exchange monitored by mass spectrometry to visualize the details of FVIIa activation by comparing the exchange kinetics of distinct molecular states, namely zymogen FVII, endoproteolytically cleaved FVIIa, TFbound zymogen FVII, TF-bound FVIIa, and FVIIa in complex with an active site inhibitor. The hydrogen exchange kinetics of zymogen FVII and FVIIa are identical indicating highly similar solution structures. However, upon tissue factor binding, FVIIa undergoes dramatic structural stabilization as indicated by decreased exchange rates localized throughout the protease domain and in distant parts of the light chain, spanning across 50 Å and revealing a concerted interplay between functional sites in FVIIa. The results provide novel insights into the cofactor-induced activation of this important protease and reveal the potential for allosteric regulation in the trypsin family of proteases. Coagulation factor VII (FVII)2 circulates in the blood with ϳ1% in a two-chain form (FVIIa) and the remainder as singlechain zymogen FVII. FVIIa consists of a trypsin-like protease domain and an N-terminal light chain composed of a membrane-binding ␥-carboxyglutamate-rich domain (Gla domain) and two epidermal growth factor-like domains (EGF1 and EGF2) (1). Upon tissue injury, tissue factor (TF) becomes exposed, and the TF⅐FVIIa complex forms and serves as the initiator of the blood coagulation cascade (2). Numerous coagulation proteases function optimally only when complexed to cofactors, and substantial biochemical evidence supports the concept that several of these cofactors induce allosteric changes in the conformation of their cognate enzymes (1, 2). TF functions to localize FVIIa and as an allosteric regulator, which dramatically enhances the activity of FVIIa. Similar to the trypsinogen-trypsin pair, zymogen FVII is converted to FVIIa by proteolysis of an internal peptide bond. A canonical "activation domain" was defined in trypsin, which includes the N terminus created upon endoproteolytic activation and three loops referred to as the activation loops (3). In trypsin, the newly generated N-terminal tail spontaneously inserts itself into a cavity close to the three activation loops, termed the activation pocket, resulting in the formation of a critical salt bridge between the N-terminal Ile-16 and Asp-194 of the active site or and in FVIIa (the chymotrypsin numbering is denoted in superscript with parentheses). This salt bridge leads to the formation of a correctly assembled S1 pocket of the active site and full activity. FVIIa, however, has very low activity following endoproteolytic activation and does not spontaneously rearrange into the active form. The three-dimensional structure of FVIIa bound to TF has been solved providing important structural details of the complex and the active form of FVIIa (4). In the complex, parts of the light chain a...

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Section: Discussionmentioning

confidence: 83%

Allosteric Activation of Coagulation Factor VIIa Visualized by Hydrogen Exchange

Rand

Jørgensen

Olsen

et al. 2006

Journal of Biological Chemistry

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“…The effect on the amidolytic activity indicates that Ca 2ϩ -binding to EGF1 causes a reorientation of the Gla domain relative to EGF1 and that the ensuing conformational change is propagated by alterations to the inter-EGF1-EGF2 angle and thence the hydrophobic interface between EGF2 and the serine protease domain. Similar conformational transitions occur between the N-terminal EGF domain and the serine protease domain in FVII (20).…”

mentioning

confidence: 86%

The N-terminal Epidermal Growth Factor-like Domain of Coagulation Factor IX

Persson

Villoutreix

Thämlitz³

et al. 2002

Journal of Biological Chemistry

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The absence or reduced activity of coagulation factor IX (FIX) causes the severe bleeding disorder hemophilia B. FIX contains an N-terminal Gla domain followed by two epidermal growth factor-like (EGF) domains and a serine protease domain. In this study, the epitope of monoclonal antibody AW, which is directed against the C-terminal part of the first EGF domain in human FIX, was defined, and the antibody was used to study interactions between the EGF domain of FIX and other coagulation proteins. Antibody AW completely blocks activation of FIX by activated factor XI, but activation by activated factor FVII-tissue factor is inhibited only slightly. The antibody also causes a marginal reduction in the apparent k cat for factor X both in the presence and absence of activated factor VIII. Based on these results, we produced a preliminary model of the structure of the activated factor IX-activated factor VIII-AW complex on the surface of phospholipid. The model suggests that in the Xase complex, EGF1 of activated factor IX is not involved in direct binding to activated factor VIII. Studies of the interaction of antibody AW with a mutated FIX molecule (R94D) also suggest that the Glu 78 -Arg 94 salt bridge is not important for maintaining the structure of FIX.

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“…The considerable distance between the interface between TF and the protease domain of FVIIa and the active site allows for a multitude of possible allosteric pathways leading to the active state. Interestingly, conformational changes associated with locking FVIIa in the active state appear to spread as far as to the first epidermal growth factor-like domain (10). Nevertheless, two specific amino acid replacements in FVIIa have recently been shown to yield variants with increased intrinsic (TF-independent) activity.…”

mentioning

confidence: 99%

Rational design of coagulation factor VIIa variants with substantially increased intrinsic activity

Persson

Kjalke²,

Olsen³

2001

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

117

218

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A trace amount of coagulation factor VII (FVII) circulates in the blood in the activated form, FVIIa (EC 3.4.21.21), formed by internal proteolysis. To avoid disseminated thrombus formation, FVIIa remains in a conformation with zymogen-like properties. Association with tissue factor (TF), locally exposed upon vascular injury, is necessary to render FVIIa biologically active and initiate blood clotting. We have designed potent mutants of FVIIa by replacing residues believed to function as determinants for the inherent zymogenicity. The TF-independent rate of factor X activation was dramatically improved, up to about 100-fold faster than that obtained with the wild-type enzyme and close to that of the FVIIa-soluble TF complex. The mutants appear to retain the substrate specificity of the parent enzyme and can be further stimulated by TF. Insights into the mechanism behind the increased activity of the mutants, presumably also pertinent to the TFinduced, allosteric stimulation of FVIIa activity, were obtained by studying their calcium dependence and the accessibility of the N terminus of the protease domain to chemical modification. The FVIIa analogues promise to offer a more efficacious treatment of bleeding episodes especially in hemophiliacs with inhibitory antibodies precluding conventional replacement therapy.

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Activation and Active Site Occupation Alter Conformation in the Region of the First Epidermal Growth Factor-like Domain of Human Factor VII

Cited by 19 publications

References 44 publications

Allosteric Activation of Coagulation Factor VIIa Visualized by Hydrogen Exchange

Allosteric Activation of Coagulation Factor VIIa Visualized by Hydrogen Exchange

The N-terminal Epidermal Growth Factor-like Domain of Coagulation Factor IX

Rational design of coagulation factor VIIa variants with substantially increased intrinsic activity

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