Flooding Enzymes: Quantifying the Contributions ofInterstitial Water and Cavity Shape to Ligand Binding Using ExtendedLinear Response Free Energy Calculations

摘要

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 moietyof BISA and moieties in the back of the enzyme active site, as wellas to probe the energetic importance of the potentially large numberof interstitial waters contacted by the BISA scaffold, we have designedseveral mutants of Asn75. GR-N75A removes a key hydrogen bond donorto the sulfonate of BISA, but also serves to introduce an additionalinterstitial water, due to the newly created space of the mutation.GR- N75L should also show the loss of a hydrogen bond donor to thesulfonate of BISA, but does not (a priori) seem to permit an additionalinterstitial water contact. In order to investigate the dynamics,structure, and energies of this water-mediated complexation, we haveemployed the extended linear response (ELR) approach for the calculationof binding free energies to GR, using the YASARA2 knowledge basedforce field on a set of ten GR complexes, and yielding an R-squaredvalue of 0.85 and a RMSE of 2.0 kJ/mol. Surprisingly, the inhibitorset produces a uniformly large interstitial water contribution tothe electrostatic interaction energy (⟨V^el⟩), ranging from 30 to >50%, except for the naturalsubstrate (d-glutamate), which has only a 7% contributionof ⟨V^el⟩ from water. Thebroader implications for predicting and exploiting significant interstitialwater contacts in ligand–enzyme complexation are discussed.