Abstract
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.
Highlights
Thrombin is a serine proteinase that plays a fundamental role in several important physiological and pathological processes, such as the coagulation, anticoagulation and fibrinolytic pathways, and is becoming an important target for drug design
A Guinier analysis of Small-angle X-ray scattering (SAXS) data [16] provides the radius of gyration, which is similar for both aTh and aTh-Phe-Pro-Arg-chloromethyl ketone (PPACK), of about 23 A (Fig. 1B; Table 1)
To further monitor the conformational changes of aTh and aTh-PPACK by molecular dynamics simulations (MD), we separately evaluated the root mean square deviation (RMSD) of the simulated systems using the crystallographic structure in L-chain (Fig. 5A) and H-chain (Fig. 5B) as a reference
Summary
Thrombin is a serine proteinase that plays a fundamental role in several important physiological and pathological processes, such as the coagulation, anticoagulation and fibrinolytic pathways, and is becoming an important target for drug design. The crystal structure of aTh was elucidated more than 20 years ago [1]; since more than 300 crystal structures have been reported so far, including complexes with various ligands, structures solved under varying chemical conditions such as pH, precipitants and coadjuvants, mutants designed to abrogate the autoproteolytic degradation [2,3] and the wild-type recombinant human aTh in the absence of Na+ ions [4]. From this large database, no large conformational changes are observed [5]. Despite the wealth of structures available to date, there is no consolidated structural study in solution of wild-type human aTh in the absence of inhibitors
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