Flooding enzymes: quantifying the contributions of interstitial water and cavity shape to ligand binding using extended linear response free energy calculations.
Flooding enzymes: quantifying the contributions of interstitial water and cavity shape to ligand binding using extended linear response free energy calculations.
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DOI：
10.1021/ci400244x
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发表时间：
2013-09-23
影响因子：
5.6
通讯作者：
Spies MA
中科院分区：
文献类型：
--
作者：
Whalen KL;Spies MA
谷氨酸消旋酶(GR)是一种不依赖辅因子的氨基酸消旋酶，近年来作为抗菌药物的靶标受到越来越多的关注。GR有许多高分辨率的晶体结构，但这些结构总是与d-谷氨酸或非常弱的氧基盐结合。最近在硅胶筛选中发现了一些新的竞争性缓蚀剂支架，它们不是基于d-Glu，但利用了许多相同的氢键给体位置。对1-H-苯并咪唑-2-磺酸(BISA)的电子研究表明，磺酸指向GR活性中心的背面，位于最深处，类似于GR-d-谷氨酸络合物中的C2-羧酸结合位置。此外，BISA已被证明是最强的非氨基酸竞争性抑制物。以前发表的计算研究表明，这种结合强度的一部分来自与一个更封闭的活性中心的络合作用，相对于较弱的配体，其中内部水网络与主体溶剂更隔离。为了验证埋藏的BISA的磺酸盐部分和酶活性部位后面的部分之间的关键联系，以及探索BISA支架可能接触的大量间隙水的能量重要性，我们设计了几个Asn75的突变体。GR-N75A去除了BISA磺酸盐的关键氢键供体，但由于突变的新空间，还用于引入额外的间隙水。GR-N75L还应显示BISA的磺酸盐失去了氢键供体，但(先验地)似乎不允许额外的间隙水接触。为了研究这种水介导的络合反应的动力学、结构和能量，我们用扩展的线性响应(ELR)方法计算了GR的结合自由能，在一组10个GR络合物上使用了基于YASARA2知识的力场，得到R的平方为0.85，RMSE为2.0kJ/mol。令人惊讶的是，除了天然底物(d-谷氨酸)外，抑制剂组产生均匀较大的间隙水对静电相互作用能(⟨VEL⟩)的贡献，范围从30%到>；50%，它只有7%的⟨VEL⟩来自水。讨论了在配基-酶络合中预测和开发重要的间隙水接触的更广泛的意义。
Glutamate racemase (GR) is a cofactor independent amino acid racemase that has recently garnered increasing attention as an antimicrobial drug target. There are numerous high resolution crystal structures of GR, yet these are invariably bound to either d-glutamate or very weakly bound oxygen-based salts. Recent in silico screens have identified a number of new competitive inhibitor scaffolds, which are not based on d-Glu, but exploit many of the same hydrogen bond donor positions. In silico studies on 1-H-benzimidazole-2-sulfonic acid (BISA) show that the sulfonic acid points to the back of the GR active site, in the most buried region, analogous to the C2-carboxylate binding position in the GR-d-glutamate complex. Furthermore, BISA has been shown to be the strongest nonamino acid competitive inhibitor. Previously published computational studies have suggested that a portion of this binding strength is derived from complexation with a more closed active site, relative to weaker ligands, and in which the internal water network is more isolated from the bulk solvent. In order to validate key contacts between the buried sulfonate moiety of BISA and moieties in the back of the enzyme active site, as well as to probe the energetic importance of the potentially large number of interstitial waters contacted by the BISA scaffold, we have designed several mutants of Asn75. GR-N75A removes a key hydrogen bond donor to the sulfonate of BISA, but also serves to introduce an additional interstitial water, due to the newly created space of the mutation. GR- N75L should also show the loss of a hydrogen bond donor to the sulfonate of BISA, but does not (a priori) seem to permit an additional interstitial water contact. In order to investigate the dynamics, structure, and energies of this water-mediated complexation, we have employed the extended linear response (ELR) approach for the calculation of binding free energies to GR, using the YASARA2 knowledge based force field on a set of ten GR complexes, and yielding an R-squared value of 0.85 and a RMSE of 2.0 kJ/mol. Surprisingly, the inhibitor set produces a uniformly large interstitial water contribution to the electrostatic interaction energy (⟨Vel⟩), ranging from 30 to >50%, except for the natural substrate (d-glutamate), which has only a 7% contribution of ⟨Vel⟩ from water. The broader implications for predicting and exploiting significant interstitial water contacts in ligand–enzyme complexation are discussed.
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