Understanding Binding Affinity:  A Combined Isothermal Titration Calorimetry/Molecular Dynamics Study of the Binding of a Series of Hydrophobically Modified Benzamidinium Chloride Inhibitors to Trypsin

Abstract

The binding of a series of p-alkylbenzamidinium chloride inhibitors to the serine proteinase trypsin over a range of temperatures has been studied using isothermal titration (micro)calorimetry and molecular dynamics simulation techniques. The inhibitors have small structural variations at the para position of the benzamidinium ion. They show small differences in relative binding affinity but large compensating differences in enthalpy and entropy. Binding affinity decreases with increased branching at the first carbon but increases with increasing the length of a linear alkyl substituent, suggesting that steric hindrance and hydrophobic interactions play dominant roles in binding. Structural analysis showed that the backbone of the enzyme was unaffected by the change of the para substituent. In addition, binding does not correlate strongly with octanol/water partition data. To further characterize this system, the change in the heat capacity on binding, the change in solvent-accessible surface area on binding, the effect of inhibitor binding on the hydration of the active site, the pK(a) of His57, and interactions within the catalytic triad have been investigated. Although the changes in inhibitor structure are small, it is demonstrated that simple concepts such as steric hindrance, hydrophobicity, and buried surface area are insufficient to explain the binding data. Other factors, such as access to the binding site and the cost of dehydration of the active site, are of equal or greater importance.

Is it the shape of the cavity, or the shape of the water in the cavity?

“…Figure 10 compares the thermodynamics of binding (ΔJ o bind , J = G, H, S) for three series of ligands whose hydrophobic alkyl chains (i.e., the "hydrophobic tail") are increased in size by a single methylene group: i) modified arylsulfonamides to human carbonic anhydrase, HCA; [168] ii) normal alcohols to major urinary protein, MUP; [154] and iii) modified benzamidinium chlorides to trypsin. [170,171] We have also included the octanol-water partitioning data for the normal alcohols to illustrate relationships between trends of protein-ligand binding and octanol-water partitioning. HCA [168], modified modified benzamidinium chlorides to trypsin [170,171], and normal alcohols to MUP [154] are plotted against the number of methylene groups in the "tail" of each ligand.…”

“…[170,171] We have also included the octanol-water partitioning data for the normal alcohols to illustrate relationships between trends of protein-ligand binding and octanol-water partitioning. HCA [168], modified modified benzamidinium chlorides to trypsin [170,171], and normal alcohols to MUP [154] are plotted against the number of methylene groups in the "tail" of each ligand. Data from the partitioning of normal alcohols [147], between octanol and water, are also plotted against the number of methylene groups.…”

Entropy–enthalpy transduction caused by conformational shifts can obscure the forces driving protein–ligand binding

“…Moreover, information on changes in entropy and enthalpy can usefully guide the design of improved drug molecules (1), with advantageous specificity (2) and physical properties (3). However, calorimetric studies of biomolecular binding and folding often reveal unexpected changes in entropy and enthalpy that are difficult to interpret in terms of physical driving forces (4)(5)(6)(7)(8). Some of these puzzling results are instances of entropy-enthalpy compensation (9), a common but not universal (10,11) phenomenon in which perturbations of a system that increase the enthalpy also increase the entropy or vice versa; therefore, the net change in the free energy remains small.…”

Thermodynamics of Supramolecular Structure Formation in Water

“…We then discuss the binding of the alkyl group of p-alkylbenzamidinium chloride to trypsin, (Talhout et al, 2003). Molecules with an amidinium group (-C(NH 2 )=NH 2 + ) are known to be an inhibitor of trypsin.…”

“…Thermodynamic parameters of binding of alkyl groups to proteins also showed very diverse compensation temperatures, indicating that hydrophobic interactions are characterized by the non-compensation effects of enthalpy and entropy. The findings are important to interpret the thermodynamic data of binding of biological interest, and also to rational design of drugs based on protein structures (Talhout et al, 2003;Malham et al, 2005).…”