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首页> 外文期刊>Biochemistry >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
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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

机译：有序水的熵损失说明了抗生素新霉素与DNA促旋酶抗性突变体的结合较弱：热力学和晶体学研究

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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.
机译：新霉素是一种与DNA促旋酶B亚基的24 kDa片段结合的抗生素。天然存在的耐药性是由Arg-136突变引起的，该突变与氢结合到新霉素的香豆素环上。我们已应用量热法来表征新霉素与野生型和R136H突变型24 kDa片段的结合。突变后，Kd在300 K时从32 nM增加到1200 nM。结合焓对突变体更有利（DeltaH度从-12.1转变为-17.5 kcal / mol），而结合熵则较差（ TDeltaS度从-1.8更改为-9.4 kcal / mol。如果Arg残基的丢失减少了氢键，则这两个变化都与预期的方向相反。结合时恒定压力下的热容变化（DeltaCp）从-295到-454 cal mol-1 K-1。我们还报告了R136H 24 kDa片段和新霉素之间的复合物的晶体结构，分辨率为2.3A。尽管DeltaCp的变化通常可以解释为反映了结合时疏水表面的埋藏增加，但该结构显示出很小的下降。此外，有序的水分子被螯合到通过去除胍基而腾空的体积中。将测得的热力学参数与使用经验关系从结构数据估计的热力学参数进行比较时，存在很大差异。这些差异似乎是由于络合时螯合有序水分子的作用而引起的。将新霉素与突变蛋白连接的水介导的氢键起了良好的焓贡献，而水的固定化导致熵成本和系统热容量的降低。这种对DeltaCp，DeltaH度和TDeltaS度的负面影响似乎是水分子的一般特性，当配体与蛋白质结合时，水分子就会被隔离。

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