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Binding of Zn 2+ to a Ca 2+ loop allosterically attenuates the activity of factor VIIa and reduces its affinity for tissue factor
Author(s) -
Petersen Lars C.,
Ård PerOla Freskg,
Persson Egon,
Olsen Ole H.,
Nielsen Lars S.
Publication year - 2000
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1110/ps.9.5.859
Subject(s) - allosteric regulation , chemistry , factor x , serine protease , tissue factor , binding site , proteases , cofactor , stereochemistry , protease , biochemistry , biophysics , enzyme , coagulation , thrombin , biology , platelet , immunology , psychology , psychiatry
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 Zn 2+ binding to FVIIa in the free form and in complex with TF were investigated. Equilibrium dialysis suggested that two Zn 2+ bind with high affinity to FVIIa outside the N‐terminal y‐carboxyglutamic acid (Gla) domain. Binding of Zn 2+ to FVIIa, which was influenced by the presence of Ca 2+ , 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 Zn 2+ inhibition. A search for putative Zn 2+ binding sites in the crystal structure of the FVIIa protease domain was performed by Grid calculations. We identified a pair of Zn 2+ binding sites in the Glu210–Glu220 Ca 2+ 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 Zn 2+ ‐mediated allosteric down‐regulation of FVIIa's activity.