Novobiocin is an antibiotic which binds to a 24 kDa fragment fromthe B subunit of DNAgyrase. Naturally occurring resistance arises from mutation ofArg-136 which hydrogen bonds to thecoumarin ring of novobiocin. We have applied calorimetry tocharacterize the binding of novobiocin towild-type and R136H mutant 24 kDa fragments. Upon mutation, the
Kd increases from 32 to 1200 nMat 300 K. The enthalpy of binding is more favorable for the mutant(
H shifts from -12.1 to -17.5kcal/mol), and the entropy of binding is much less favorable(
TS changes from -1.8 to -9.4kcal/mol). Both of these changes are in the direction opposite to thatexpected if the loss of the Arg residuereduces hydrogen bonding. The change in heat capacity at constantpressure upon binding (
Cp)shiftsfrom -295 to -454 cal mol
-1K
-1. We also report the crystalstructure, at 2.3 Å resolution, of a complexbetween the R136H 24 kDa fragment and novobiocin. Although thechange in
Cp often wouldbeinterpreted as reflecting increased burial of hydrophobic surface onbinding, this structure reveals a smalldecrease. Furthermore, an ordered water molecule is sequesteredinto the volume vacated by removal ofthe guanidinium group. There are large discrepancies when themeasured thermodynamic parameters arecompared to those estimated from the structural data using empiricalrelationships. These differencesseem to arise from the effects of sequestering ordered water moleculesupon complexation. The water-mediated hydrogen bonds linking novobiocin to the mutant protein make afavorable enthalpic contribution,whereas the immobilization of the water leads to an entropic cost and areduction in the heat capacity ofthe system. Such a negative contribution to
Cp,
H, and
TS appears to be a general propertyofwater molecules that are sequestered when ligands bind toproteins.