Thermodynamic compensation upon binding to exosite 1 and the active site of thrombin.

Several lines of experimental evidence including amide exchange and NMR suggest that ligands binding to thrombin cause reduced backbone dynamics. Binding of the covalent inhibitor dPhe-Pro-Arg chloromethyl ketone to the active site serine, as well as noncovalent binding of a fragment of the regulatory protein, thrombomodulin, to exosite 1 on the back side of the thrombin molecule both cause reduced dynamics. However, the reduced dynamics do not appear to be accompanied by significant conformational changes. In addition, binding of ligands to the active site does not change the affinity of thrombomodulin fragments binding to exosite 1; however, the thermodynamic coupling between exosite 1 and the active site has not been fully explored. We present isothermal titration calorimetry experiments that probe changes in enthalpy and entropy upon formation of binary ligand complexes. The approach relies on stringent thrombin preparation methods and on the use of dansyl-l-arginine-(3-methyl-1,5-pantanediyl)amide and a DNA aptamer as ligands with ideal thermodynamic signatures for binding to the active site and to exosite 1. Using this approach, the binding thermodynamic signatures of each ligand alone as well as the binding signatures of each ligand when the other binding site was occupied were measured. Different exosite 1 ligands with widely varied thermodynamic signatures cause a similar reduction in ΔH and a concomitantly lower entropy cost upon DAPA binding at the active site. The results suggest a general phenomenon of enthalpy-entropy compensation consistent with reduction of dynamics/increased folding of thrombin upon ligand binding to either the active site or exosite 1.

Mutations in the fourth EGF-like domain affect thrombomodulin-induced changes in the active site of thrombin.

A number of alanine and more conservative mutants of residues in the fourth domain of thrombomodulin (TM) were prepared and assayed for protein C activation and for thrombin binding. Several of the alanine mutations appeared to cause misfolding or structural defects as assessed by poor expression and/or NMR HSQC experiments, while more conservative mutations at the same site appeared to allow correct folding and preserved activity. Several of the conservative mutants bound more weakly to thrombin despite the fact that the fourth domain does not directly contact thrombin in the crystal structure of the thrombin-TM complex. A few of the mutant TM fragments bound thrombin with an affinity similar to that of the wild type but exhibited decreases in k cat for protein C activation. These mutants were also less able to cause a change in the steady state fluorescence of fluorescein-EGR-chloromethylketone bound to the active site of thrombin. These results suggest that some residues within the fourth domain of TM may primarily interact with protein C but others are functionally important for altering the way TM interacts with thrombin. Residues in the fourth domain that primarily affect k cat for protein C activation may do this by changing the active site of thrombin.