Civera Monica, Sattin Sara
Department of Chemistry, Università Degli Studi di Milano, Milan, Italy.
Front Mol Biosci. 2021 Feb 3;8:628596. doi: 10.3389/fmolb.2021.628596. eCollection 2021.
Bacteria have developed different bet hedging strategies to survive hostile environments and stressful conditions with persistency being maybe the most elegant yet still poorly understood one. Persisters' temporary tolerance to antibiotic treatment hints at their role not only in chronic and recurrent infections but also in the insurgence of resistant strains. Therefore, hampering persisters formation might represent an innovative strategy in the quest for new effective antimicrobial compounds. Among the molecular mechanisms postulated for the persister phenotypic switch, we decided to focus our attention on the stringent response and, in particular, on the upstream triggering step that is the accumulation of guanosine tetra- and pentaphosphate, collectivity called (p)ppGpp. Intracellular levels of (p)ppGpp are regulated by a superfamily of enzymes called RSH (RelA/SpoT homologue) that are able to promote its synthesis pyrophosphate transfer from an ATP molecule to the 3' position of either GDP or GTP. These enzymes are classified based on the structural domain(s) present (only synthetase, only hydrolase, or both). Here we present our work on Rel (from ), still the only bifunctional Rel protein for which a GDP-bound "synthetase-ON" structure is available. Analysis of the synthetase site, occupied only by GDP, revealed a partially active state, where the supposed ATP binding region is not conformationally apt to accommodate it. In order to achieve a protein model that gets closer to a fully active state, we generated a chimera structure of Rel by homology modeling, starting from the crystal structure of the catalytically competent state of RelP, a smaller, single-domain, Rel protein from . Molecular dynamics simulations allowed verifying the stability of the generated chimera model. Virtual screening and ligand design studies are underway.
细菌已经发展出不同的应急策略来在恶劣环境和压力条件下生存，而持续性或许是其中最巧妙但仍知之甚少的一种。持留菌对抗生素治疗的暂时耐受性表明它们不仅在慢性和复发性感染中起作用，而且在耐药菌株的出现中也起作用。因此，阻碍持留菌的形成可能是寻找新型有效抗菌化合物的一种创新策略。在为持留菌表型转换所假定的分子机制中，我们决定将注意力集中在严谨反应上，特别是集中在其上游触发步骤，即四磷酸鸟苷和五磷酸鸟苷的积累，统称为（p）ppGpp。（p）ppGpp的细胞内水平由一类称为RSH（RelA/SpoT同源物）的酶超家族调节，这些酶能够通过将焦磷酸从ATP分子转移到GDP或GTP的3'位置来促进其合成。这些酶根据存在的结构域进行分类（仅合成酶、仅水解酶或两者皆有）。在此，我们展示了关于Rel（来自）的研究工作，它仍然是唯一一种有GDP结合的“合成酶开启”结构的双功能Rel蛋白。对仅由GDP占据的合成酶位点的分析揭示了一种部分活性状态，其中假定的ATP结合区域在构象上不适合容纳它。为了获得一个更接近完全活性状态的蛋白质模型，我们通过同源建模生成了Rel的嵌合体结构，起始于RelP催化活性状态的晶体结构，RelP是一种来自的较小的单结构域Rel蛋白。分子动力学模拟验证了所生成嵌合体模型的稳定性。虚拟筛选和配体设计研究正在进行中。
