The Entropic Penalty of Ordered Water Accounts for Weaker Binding of the Antibiotic Novobiocin to a Resistant Mutant of DNA Gyrase:  A Thermodynamic and Crystallographic Study

Abstract

Novobiocin is an antibiotic which binds to a 24 kDa fragment from the B subunit of DNA gyrase. Naturally occurring resistance arises from mutation of Arg-136 which hydrogen bonds to the coumarin ring of novobiocin. We have applied calorimetry to characterize the binding of novobiocin to wild-type and R136H mutant 24 kDa fragments. Upon mutation, the Kd increases from 32 to 1200 nM at 300 K. The enthalpy of binding is more favorable for the mutant (DeltaH degrees shifts from -12.1 to -17.5 kcal/mol), and the entropy of binding is much less favorable (TDeltaS degrees changes from -1.8 to -9.4 kcal/mol). Both of these changes are in the direction opposite to that expected if the loss of the Arg residue reduces hydrogen bonding. The change in heat capacity at constant pressure upon binding (DeltaCp) shifts from -295 to -454 cal mol-1 K-1. We also report the crystal structure, at 2.3 A resolution, of a complex between the R136H 24 kDa fragment and novobiocin. Although the change in DeltaCp often would be interpreted as reflecting increased burial of hydrophobic surface on binding, this structure reveals a small decrease. Furthermore, an ordered water molecule is sequestered into the volume vacated by removal of the guanidinium group. There are large discrepancies when the measured thermodynamic parameters are compared to those estimated from the structural data using empirical relationships. These differences seem to arise from the effects of sequestering ordered water molecules upon complexation. The water-mediated hydrogen bonds linking novobiocin to the mutant protein make a favorable enthalpic contribution, whereas the immobilization of the water leads to an entropic cost and a reduction in the heat capacity of the system. Such a negative contribution to DeltaCp, DeltaH degrees , and TDeltaS degrees appears to be a general property of water molecules that are sequestered when ligands bind to proteins.

Structural mechanism for affinity maturation of an anti-lysozyme antibody

“…In comparable protein͞protein interactions, mutations that increase enthalpies of reaction may also contribute to unfavorable entropies (enthalpy-entropy compensation). The negative change in entropy after F10.6.6͞HEL complex formation, as compared with the neutral change in entropy for the D44.1͞HEL reaction, can be explained in part by the extra water molecule buried at the interface (39). Other possible causes for this effect include desolvation of a larger surface of interaction and differences in the conformation of uncomplexed and complexed antibody and antigen.…”

Gaining ligand selectivity in thyroid hormone receptors via entropy

“…It is clear that such effects play important roles in several protein-ligand interactions (18,19); examples include improved inhibitor binding (20,21) and many other aspects of protein/small molecule interactions, such as ligand dissociation paths (22) and ion permeation (23). It is interesting to evaluate previous NR structures in the light of our results.…”

A Balancing Act between Net Uptake of Water during Dihydrofolate Binding and Net Release of Water upon NADPH Binding in R67 Dihydrofolate Reductase

“…Dunitz (58) has proposed that enthalpy-entropy compensation commonly occurs in weak interactions. Also this type of compensation is a hallmark of water involvement (80,81) as calculations indicate that the ⌬H value associated with cavity formation for accommodating a solvent molecule is exactly balanced by the entropy of the cavity (82,83). Thus, contributions to ⌬H and ⌬S can occur but will not necessarily show up in ⌬G as ⌬G solvation equals zero (ϭ ⌬H solvation Ϫ T⌬S solvation ).…”