Abstract
The conformational properties of trypsin-like proteases and their zymogen forms remain controversial because of a lack of sufficient information on their free forms. Specifically, it is unclear whether the free protease is zymogen-like and shifts to its mature form upon a ligand-induced fit or exists in multiple conformations in equilibrium from which the ligand selects the optimal fit via conformational selection. Here we report the results of 19F NMR measurements that reveal the conformational properties of a protease and its zymogen precursor in the free form. Using the trypsin-like, clotting protease thrombin as a relevant model system, we show that its conformation is quite different from that of its direct zymogen precursor prethrombin-2 and more similar to that of its fully active Na+-bound form. The results cast doubts on recent hypotheses that free thrombin is zymogen-like and transitions to protease-like forms upon ligand binding. Rather, they validate the scenario emerged from previous findings of X-ray crystallography and rapid kinetics supporting a pre-existing equilibrium between open (E) and closed (E*) forms of the active site. In this scenario, prethrombin-2 is more dynamic and exists predominantly in the E* form, whereas thrombin is more rigid and exists predominantly in the E form. Ligand binding to thrombin takes place exclusively in the E form without significant changes in the overall conformation. In summary, these results disclose the structural architecture of the free forms of thrombin and prethrombin-2, consistent with an E*–E equilibrium and providing no evidence that free thrombin is zymogen-like.
Highlights
The conformational properties of trypsin-like proteases and their zymogen forms remain controversial because of a lack of sufficient information on their free forms
We investigate for the first time the conformation of the protease thrombin and its zymogen precursor prethrombin-2 in the free form using 19F NMR
The 19F-labeled prethrombin-2 structure features a conformation of the active site similar to that of WT [13] (RMSD ϭ 0.33 Å)
Summary
The conformational properties of trypsin-like proteases and their zymogen forms remain controversial because of a lack of sufficient information on their free forms. Using the trypsin-like, clotting protease thrombin as a relevant model system, we show that its conformation is quite different from that of its direct zymogen precursor prethrombin-2 and more similar to that of its fully active Na؉-bound form. The results cast doubts on recent hypotheses that free thrombin is zymogen-like and transitions to protease-like forms upon ligand binding Rather, they validate the scenario emerged from previous findings of X-ray crystallography and rapid kinetics supporting a pre-existing equilibrium between open (E) and closed (E*) forms of the active site. Ligand binding to thrombin takes place exclusively in the E form without significant changes in the overall conformation These results disclose the structural architecture of the free forms of thrombin and prethrombin-2, consistent with an E*–E equilibrium and providing no evidence that free thrombin is zymogen-like. 95.0 5.0 0.0 a RMSD from ideal bond lengths and angles and RMSD in B-factors of bonded atoms. b mm, main chain–main chain; ms, main chain–side chain; ss, side chain–side chain
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