News and Publications

Allosteric Regulation of Serine Protease Domains

C.D. Dickinson, J. Shobe, W. Ruf

The catalytic activity of serine proteases is allosterically regulated. We study coagulation factor VIIa, a cofactor-dependent serine protease. As a free enzyme, factor VIIa makes an incomplete transition from zymogen to enzyme and thus remains in a conformational state with low enzymatic activity. Interaction with cofactor supports the conformational transition to the active enzyme, thus providing a mechanism for the tight control of the proteolytic function of factor VIIa in vivo. These data suggest that the function of serine proteases is regulated by a global conformational switch.

Using site-directed mutagenesis, we obtained evidence that the side chains of specific residues are involved in various conformational linkages. We found conformational linkages between functional regions of the protease domain of factor VIIa that are involved in binding to cofactors, docking to macromolecular substrate, and recognition and cleavage of scissile bonds. Studies with a conformation-sensitive monoclonal antibody to the macromolecular substrate exosite indicated that the conformational linkages between the functional regions of the VIIa protease domain are discrete. The kinetics of binding of factor VIIa to this antibody are influenced, in different ways, by cofactor interactions with the VIIa protease domain and by occupancy of the active site. We have further mapped the residue side chain in the anitbody epitope that reorients after inhibitor binding to the active site of factor VIIa and thus are beginning to unravel the conformational linkages at the molecular level. These studies provide novel insight into allosteric regulation of the function of serine proteases and fundamental information for the design of specific inhibitors of this class of prothrombotic enzymes.

Cell Biology of Tissue Factor

H.-Y. Huang, M. Riewald, I. Ott, E.G. Fischer, Y. Miyagi, B.M. Mueller, W. Ruf

Our analysis of the prometastatic functions of tissue factor (TF) indicated a cooperation of extracellular proteolytic activity and intracellular signaling functions of the TF cytoplasmic domain. Using 2-hybrid screening, we detected intracellular ligands for this domain. We cloned 3 novel proteins and are investigating their functional relationship to TF.

We also found that actin-binding protein 280 is specifically recruited upon extracellular ligation of TF. Because actin-binding protein 280 and TF were not constitutively associated, an induced-fit mechanism most likely is responsible for the interaction of the short cytoplasmic domain of TF with the actin-binding protein. The link of TF to the actin cytoskeleton suggested roles for TF in cell adhesion. Immobilized ligands for TF support cell adhesion and migration, and these cellular functions do not depend on the proteolytic activity of factor VIIa, the serine protease ligand of TF, which can elicit cellular responses through an unknown protease-activated receptor. It is unclear which extracellular ligand for TF supports the migratory function of cells. Because TF is expressed by invasive tumors and angiogenic endothelial cells, we are exploring the possibility that local vascular hyperpermeability causes the extravasation of plasma proteins that participate in macromolecular assemblies with TF to support cell migration. Studies with melanoma cells deficient in actin-binding protein 280 indicated that the protein is required for TF-dependent cell adhesion and spreading. These results provide unambiguous proof that interaction of the TF cytoplasmic domain with actin-binding protein 280 has a functional role in TF-mediated signaling. Current efforts are directed toward dissecting the intracellular signaling pathways by which TF influences the migratory functions of cells.

PUBLICATIONS

Dickinson, C.D., Shobe, J., Ruf, W. Influence of cofactor binding and active site occupancy on the conformation of the macromolecular substrate exosite of factor VIIa. J. Mol. Biol. 277:959, 1998.

Edgington, T.S., Dickinson, C.D., Ruf, W. The structural basis of function of the TF*VIIa complex in the cellular initiation of coagulation. Thromb. Haemost. 78:401, 1997.

Huang, M., Syed, R., Stura, E.A., Stone, M.J., Stefanko, R.S., Ruf, W., Edgington, T.S., Wilson, I.A. The mechanism of an inhibitory antibody on TF-initiated blood coagulation revealed by the crystal structures of human tissue factor, Fab 5G9 and TF-5G9 complex. J. Mol. Biol. 275:873, 1998.

Mueller, B.M., Ruf, W. Requirement for binding of catalytically active factor VIIa in tissue factor dependent experimental metastasis. J. Clin. Invest. 101:1372, 1998.

Ott, I., Fischer, E.G., Miyagi, Y., Mueller, B.M., Ruf, W. A role for tissue factor in cell adhesion and migration mediated by interaction with actin binding protein 280. J. Cell Biol. 140:1241, 1998.

Ruf, W. Faktor VII und Gewebethromboplastin: Der extrinsische Aktivierungskomplex. In: Hämostaseologie. Pötzsch, B., Müller-Berghaus, G. (Eds.). Springer-Verlag, New York, in press.

Ruf, W. Gewebethromboplastin: Biochemie, Molekularbiologie und Physiologie. In: Hämostaseologie. Pötzsch, B., Müller-Berghaus, G. (Eds.). Springer-Verlag, New York, in press.

Ruf, W. The interaction of factor VIIa with tissue factor: Insight into the mechanism of cofactor-mediated activation of factor VIIa. Blood Coagul. Fibrinolysis 9:S73, 1998.