Structural Studies on a Pair of Circulatory Proteins: Cofactors or “Two Cats in a Bag”?
Cynthia B. Peterson, PhD, Professor and Head, Biochemistry and Cellular and Molecular Biology, University of Tennessee, Knoxville; Director, UT/ORNL School of Genome Science and Technology
Proteases regulate many processes in biology, including blood coagulation and clot lysis, the immune response, and processes that remodel the extracellular matrix (ECM) during wound healing and cell migration. Because of the importance of proteases in such a wide realm of biology, their activity must be closely checked. This is accomplished in part by the serine protease inhibitors (serpins), which form inactive covalent complexes with their target proteases. Often accessory factors can modulate this inhibition process. Plasminogen activator inhibitor-1 (PAI-1) is the major inhibitor of both urokinase (uPA) and tissue plasminogen activator (tPA) that lyse fibrin clots and also cleave ECM components. PAI-1 forms a stable complex with these proteases in a suicide fashion. The active conformation of PAI-1 is metastable and spontaneously converts to a latent form with a half-life of 1.1 hours at 370C. The half-life of the active conformation is extended to 1.5 hours when PAI-1 is bound to the plasma protein vitronectin. The fact that vitronectin is also conformationally labile, combined with confusing observations regarding the activities of vitronectin and PAI-1 complexes that are both protease-dependent and –independent, has made studies on these proteins work on what has been called “two cats in a bag.” Nonetheless, the complex between vitronectin and PAI-1 has been characterized using analytical ultracentrifugation, mutagenesis, and surface plasmon resonance to define an extensive binding surface between the two proteins. The observed stabilization of PAi-1 by vitronectin is metal-dependent. Human PAI-1 (hPAI-1) more rapidly converts to the latent conformation in the presence of Co, Ni, and Cu. Strikingly, the half-life is much longer in the presence of vitronectin and these metals, increasing to nearly 6 hours in the presence of copper. Binding studies using stopped-flow methods and changes in intrinsic fluorescence of PAI-1 show changes in conformation that occur upon metal binding. From this work, the binding to these transition metals is in the micromolar range. This affinity is reasonable considering the physiological concentrations and bioavailability of these metals. These effects are intriguing considering the roles of metals in extracellular processes, such as neural signaling, cancer progression, and inflammation, all processes where vitronectin and PAI-1 have regulatory functions.