by Vivian de Almeira Silva, Maria Thereza Cargnelutti, Guilherme M. Giesel, Leonardo C. Palmieri, Robson Q. Monteiro, Hugo Verli, Luis Mauricio T. R. Lima
Thrombin is a serine proteinase that plays a fundamental role in coagulation. In this study, we address the effects of ligand site recognition by alpha-thrombin on conformation and energetics in solution. Active site occupation induces large changes in secondary structure content in thrombin as shown by circular dichroism. Thrombin-D-Phe-Pro-Arg-chloromethyl ketone (PPACK) exhibits enhanced equilibrium and kinetic stability compared to free thrombin, whose difference is rooted in the unfolding step. Small-angle X-ray scattering (SAXS) measurements in solution reveal an overall similarity in the molecular envelope of thrombin and thrombin-PPACK, which differs from the crystal structure of thrombin. Molecular dynamics simulations performed with thrombin lead to different conformations than the one observed in the crystal structure. These data shed light on the diversity of thrombin conformers not previously observed in crystal structures with distinguished catalytic and conformational behaviors, which might have direct implications on novel strategies to design direct thrombin inhibitors.