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Binding of Zn2+ to a Ca2+ loop allosterically attenuates the activity of factor VIIa and reduces its affinity for tissue factor

Published online by Cambridge University Press:  01 May 2000

LARS C. PETERSEN
Affiliation:
Tissue Factor/Factor VII Research, Novo Nordisk A/S, Novo Nordisk Park, DK-2760 Måløv, Denmark
OLE H. OLSEN
Affiliation:
Medicinal Chemistry Research IV, Novo Nordisk A/S, Novo Nordisk Park, DK-2760 Måløv, Denmark
LARS S. NIELSEN
Affiliation:
Molecular Genetics, Novo Nordisk A/S, Novo Alle 1, DK-2880 Bagsværd, Denmark
PER-OLA FRESKGÅRD
Affiliation:
Tissue Factor/Factor VII Research, Novo Nordisk A/S, Novo Nordisk Park, DK-2760 Måløv, Denmark
EGON PERSSON
Affiliation:
Tissue Factor/Factor VII Research, Novo Nordisk A/S, Novo Nordisk Park, DK-2760 Måløv, Denmark
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Abstract

The protease domain of coagulation factor VIIa (FVIIa) is homologous to trypsin with a similar active site architecture. The catalytic function of FVIIa is regulated by allosteric modulations induced by binding of divalent metal ions and the cofactor tissue factor (TF). To further elucidate the mechanisms behind these transformations, the effects of Zn2+ binding to FVIIa in the free form and in complex with TF were investigated. Equilibrium dialysis suggested that two Zn2+ bind with high affinity to FVIIa outside the N-terminal γ-carboxyglutamic acid (Gla) domain. Binding of Zn2+ to FVIIa, which was influenced by the presence of Ca2+, resulted in decreased amidolytic activity and slightly reduced affinity for TF. After binding to TF, FVIIa was less susceptible to zinc inhibition. Alanine substitutions for either of two histidine residues unique for FVIIa, His216, and His257, produced FVIIa variants with decreased sensitivity to Zn2+ inhibition. A search for putative Zn2+ binding sites in the crystal structure of the FVIIa protease domain was performed by Grid calculations. We identified a pair of Zn2+ binding sites in the Glu210–Glu220 Ca2+ binding loop adjacent to the so-called activation domain canonical to serine proteases. Based on our results, we propose a model that describes the conformational changes underlying the Zn2+-mediated allosteric down-regulation of FVIIa's activity.

Type
Research Article
Copyright
2000 The Protein Society

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