Protonation Changes upon Ligand Binding to Trypsin and Thrombin: Structural Interpretation Based on pKa Calculations and ITC Experiments

The protonation states of a protein and a ligand can be altered upon complex formation. Such changes can be detected experimentally by isothermal titration calorimetry (ITC). For a series of ligands binding to the serine proteases trypsin and thrombin, we previously performed an extensive ITC and crystallographic study and were able to identify protonation changes for four complexes. However, since ITC measures only the overall proton exchange, it does not provide structural insights into the functional groups involved in the proton transfer. Using Poisson–Boltzmann calculations based on our recently developed PEOE_PB charges, we compute pKa values for all complexes of our former study in order to reveal the residues with altered protonation states. The results indicate that His57, a member of the catalytic triad, is responsible for the most relevant pKa shifts leading to the experimentally detected protonation changes. This finding is in contrast to our previous assumption that the observed protonation changes occur at the carboxylic group of the ligands. The newly detected proton acceptor is used for a revised factorization of the ITC data, which is necessary whenever the protonation inventory changes upon complexation. The pKa values of complexes showing no protonation change in the ITC experiment are reliably predicted in most cases, whereas predictions of strongly coupled systems remain problematic.