Two different Ca<sup>2+</sup> ion binding sites in factor VIIa and in des(1–38) factor VIIa

Schiødt, Jakob; Harrit, Niels; Christensen, Ulla; Petersen, Lars C.

doi:10.1016/0014-5793(92)81014-d

Cited by 33 publications

(35 citation statements)

References 13 publications

(11 reference statements)

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“…VIIa The apparent Kc~ obtained from activity measurements in the absence of TF was found to be 0.05 mM close to the value obtained from fluorescence measurements [4,31], which was suggested to reflect binding of Ca 2+ ions to a site in the EGF-1 domain [4,32]. The present work shows that Ca 2+ ion binding also affects the intrinsic fluorescence of des(I-44) factor VIIa.…”

Section: Binding Of Ca E+ Zn E+ and Tb 3+ To Des Gla-factorsupporting

confidence: 81%

“…The results are listed in Table 2. As previously described [4], Tb 3+ ions binds to intact factor VIIa and the des(I-38) form with similar affinities, however, the affinity for des(I-44) factor VIIa was significantly decreased. 10 mM glycylglycine, 100 mM NaC1, pH 6.5, 25°C.…”

Section: Tb 3+ Phosphorescence Measurementssupporting

confidence: 70%

“…The change in intrinsic protein fluorescence induced by Ca 2+ ion binding was measured as described before [4]. Titrations with Ca 2+ ions were performed by adding series of small volumes (2-6/al) of aqueous CaCI 2 (0, 5 mM or 0.5 M) to 1 ml of enzyme (1/aM) in a semimiero cuvette.…”

Section: Protein Fluorescence Ca Z+ Ion Titration Experimentsmentioning

confidence: 99%

“…Tb 3+ ion phosphorescence experiments were performed as described before [4]. Spectra were recorded using a Perkin-Elmer LS-50 spectrofluorometer equipped with FLDM software.…”

Section: Tb 3+ Ion Phosphorescence Experimentsmentioning

confidence: 99%

“…Ca2+-binding sites has been identified in the Gla-domain, the first EGF domain, and in the protease domain [4,5]. Binding of Ca2+-ions to one or more of these sites appears to be essential for factor VII interaction with TF.…”

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confidence: 99%

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Involvement of the hydrophobic stack residues 39–44 of factor VII_a in tissue factor interactions

1994

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Des(l-38) factor VII, and des(l--44) factor VII, were obtained by limited proteolysis. The binding of tissue factor to these factor VII,-derivatives was assessed from its stimulation of the proteolytic activity on chromogenic oligopeptide substrates. Compared to native factor VII, (Kay = 0.6 + 0.1 riM), Tissue factor binds to des(l-38) factor VII, with a lower, but still signiticant affinity (Kw = 4.8 + 0.3 nM). The activity of des(I-44) factor VII, was only slightly stimulated by TF (KTF --200 nlVO. Binding of TF depends critically on the presence of Ca 2+ ions. Ca 2+ ions stimulated the activity of factor VIIJTF with an apparent K~ = 0.16 + 0.02 raM. Factor VII, in the absence of tissue factor was stimulated by Ca 2+ with an apparent K~ = 0.05 + 0.01 raM, and similar K~ values were obtained for the truncated derivatives of factor VIIa. Measurements of Ca2+-induced changes in intrinsic protein fluorescence suggest a conformational change. The Ca 2+ ion concentration at which this change occurred was higher for des(I-44) factor VII, (apparent Kc~ = 0.14 mM) than for des(I-38)-and native factor VII, (apparent K~ = 0.04 mM). The Tb 3+ ion luminescence technique was used to further investigate the Ca 2+ binding sites. Tb 3+ ions bound with a lower affinity to des(l-44) factor VII, than to des(1-38)-and native factor VIIa. The observed drastic decrease in affinity for tissue factor as a result of truncation of the 'hydrophobic stack' residues 39--44, suggest that this region of factor VII~ provides a structural determinant that together with other regions participates in tissue factor binding.

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Section: Binding Of Ca E+ Zn E+ and Tb 3+ To Des Gla-factorsupporting

confidence: 81%

Section: Tb 3+ Phosphorescence Measurementssupporting

confidence: 70%

Section: Protein Fluorescence Ca Z+ Ion Titration Experimentsmentioning

confidence: 99%

“…Tb 3+ ion phosphorescence experiments were performed as described before [4]. Spectra were recorded using a Perkin-Elmer LS-50 spectrofluorometer equipped with FLDM software.…”

Section: Tb 3+ Ion Phosphorescence Experimentsmentioning

confidence: 99%

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confidence: 99%

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Involvement of the hydrophobic stack residues 39–44 of factor VII_a in tissue factor interactions

1994

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Predicted solution structure of zymogen human coagulation FVII

Perera¹,

Darden²,

Pedersen³

2001

J Comput Chem

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A model solution structure for the complete tissue factor-free calcium ion-bound human zymogen FVII (residues 1-406) (FVII) has been constructed to study possible conformational changes associated with the activation process and tissue factor (TF) binding. The initial structure for the present model was constructed using the X-ray crystallographic structure of human coagulation FVIIa/TF complex bound with calcium ions (Banner et al., Nature 1996, 380, 41-46). This model was subsequently subjected to lengthy molecular dynamics simulations. The Amber force field in conjunction with the PME electrostatic summation method was employed. The estimated TF free solution structure was then compared with the currently available X-ray crystal structures of FVIIa (with or without TF, variable inhibitor bound) to estimate the restructuring of FVII due to TF binding and activation. The solution structure of the zymogen FVII in the absence of TF is predicted to be an extended domain structure similar to that of the TF-bound X-ray crystal structure. An additional extension of the serine protease (SP) domain of the zymogen above a reference lipid surface by approximately 7 A was in agreement with experiment. Significant Gla-EGF1 and EGF1-EGF2 interdomain motions in the zymogen were observed. Carbohydrate dimers attached to Ser-52 and Ser-60 did not cause restructuring in this domain. Minimal restructuring of the SP domain is found upon inference of the zymogen from the activated form. The catalytic triad residues maintain the H-bonded network while Lys-341 occupies the S1 specific site in the zymogen.

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Structural changes in factor VIIa induced by Ca²⁺ and tissue factor studied using circular dichroism spectroscopy

1996

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Factor VIIa (fVIIa) is composed of four discrete domains, a y-carboxyglutamic acid (G1a)-containing domain, two epidermal growth factor (EGF)-like domains, and a serine protease domain, all of which appear to be involved, to different extents, in an optimal interaction with tissue factor (TF). All except the second EGF-like domain contain at least one Ca2+ binding site and many properties of fVIIa, e.g., TF and phospholipid binding and amidolytic activity, are Ca2+-dependent. A CD study was performed to characterize and locate the conformational changes in fVIIa induced by Ca'+ and TF binding. In addition to intact fVIIa, derivatives lacking the Gla domain or the protease domain were used. Assignment of the Ca2+-induced changes in the far-UV region of the fVIIa spectrum to the Gla domain could be made by comparing the CD spectra obtained with these fVIla derivatives. The changes primarily appeared to reflect a Ca*+-induced ordering of a-helices existing in the apo state of fVIIa. This was corroborated by models of the apo and Ca2+ forms of fVIIa constructed on the basis of known structures of homologous proteins. Far-UV spectra of the Gla domain of fVIIa, obtained as difference spectra between fVIIa derivatives, were very similar to those of isolated Gla peptides from other vitamin K-dependent plasma proteins. The near-UV CD spectrum of fVIIa was dominated by aromatic residues residing in the protease domain and specific bands affected by Ca2+ were indicative of tertiary structural alterations. The formation of a NIIa:TF complex led to secondary structural changes that appeared to be restricted to the catalytic domain, possibly shedding light on the mechanism by which T F induces an enhancement of fVIIa catalytic activity.

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Two different Ca²⁺ ion binding sites in factor VIIa and in des(1–38) factor VIIa

Cited by 33 publications

References 13 publications

Involvement of the hydrophobic stack residues 39–44 of factor VII_a in tissue factor interactions

Involvement of the hydrophobic stack residues 39–44 of factor VII_a in tissue factor interactions

Predicted solution structure of zymogen human coagulation FVII

Structural changes in factor VIIa induced by Ca²⁺ and tissue factor studied using circular dichroism spectroscopy

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Two different Ca2+ ion binding sites in factor VIIa and in des(1–38) factor VIIa

Cited by 33 publications

References 13 publications

Involvement of the hydrophobic stack residues 39–44 of factor VIIa in tissue factor interactions

Involvement of the hydrophobic stack residues 39–44 of factor VIIa in tissue factor interactions

Predicted solution structure of zymogen human coagulation FVII

Structural changes in factor VIIa induced by Ca2+ and tissue factor studied using circular dichroism spectroscopy

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Two different Ca²⁺ ion binding sites in factor VIIa and in des(1–38) factor VIIa

Involvement of the hydrophobic stack residues 39–44 of factor VII_a in tissue factor interactions

Involvement of the hydrophobic stack residues 39–44 of factor VII_a in tissue factor interactions

Structural changes in factor VIIa induced by Ca²⁺ and tissue factor studied using circular dichroism spectroscopy