Protonation changes upon ligand binding to trypsin and thrombin: structural interpretation based on pK(a)...

Area of Science:

• Biochemistry
• Structural Biology
• Computational Chemistry

Background:

• Protein-ligand complex formation can alter protonation states.
• Isothermal titration calorimetry (ITC) detects these proton exchange changes but lacks structural detail.
• Previous studies identified protonation changes in serine protease-ligand complexes but not the specific residues involved.

Purpose of the Study:

• To computationally identify residues with altered protonation states in protein-ligand complexes.
• To elucidate the structural basis for experimentally observed protonation changes.
• To refine the interpretation of ITC data by accounting for altered protonation.

Main Methods:

• Poisson-Boltzmann (PB) calculations using PEOE_PB charges.
• Computation of pK(a) values for protein-ligand complexes.
• Comparison of calculated pK(a) shifts with experimental ITC data.

Main Results:

• Calculated pK(a) values revealed His57 in the catalytic triad as the primary site of protonation changes.
• This contradicts the prior assumption that ligand carboxylic groups were responsible.
• The revised understanding of proton transfer improved the factorization of ITC data for some complexes.

Conclusions:

• His57 is identified as the key residue responsible for experimentally detected protonation changes in serine protease-ligand interactions.
• The study provides a structural explanation for ITC-observed proton exchange.
• Accurate pK(a) prediction is reliable for less coupled systems but remains challenging for strongly coupled ones.