Structural and molecular dynamics of Mycobacterium tuberculosis malic enzyme, a potential anti-TB drug target
Structural and molecular dynamics of Mycobacterium tuberculosis malic enzyme, a potential anti-TB drug target
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DOI：
10.1101/2020.07.07.192161
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发表时间：
2020-07
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通讯作者：
K. Burley;B. Cuthbert;Piyali Basu;J. Newcombe;E. M. Irimpan;Robert Quechol;Ilona P. Foik;D. Mobley;D. Beste;C. Goulding
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作者：
K. Burley;B. Cuthbert;Piyali Basu;J. Newcombe;E. M. Irimpan;Robert Quechol;Ilona P. Foik;D. Mobley;D. Beste;C. Goulding
结核病（TB）是世界范围内最致命的细菌性传染病。众所周知，它很难治疗，需要数月的抗生素鸡尾酒疗法。结核病病原体结核分枝杆菌（Mtb）的致密蜡质外膜是阻止抗生素摄入的强大屏障。随后，参与维持Mtb细胞壁完整性的酶是有希望的药物靶标。最近，我们证明了Mtb缺乏苹果酸酶（MEZ）改变了细胞壁脂质组成，并减弱了巨噬细胞的摄取。这些结果表明，MEZ为脂质生物合成提供了所需的还原能力。在这里，我们提出了MEZ的X-射线晶体结构的3.6倍分辨率，并与原核和真核苹果酸酶的已知结构进行比较。我们使用生化测定来确定其寡聚状态，并评估其动力学和热稳定性的pH值和变构调节剂的影响。为了评估MEZ与其底物苹果酸和辅因子Mn 2+和NAD（P）+之间的相互作用，我们进行了一系列分子动力学（MD）模拟。首先，MD分析证实了我们的经验观察，MEZ是异常无序的，即使加入底物和辅因子也会持续存在。第二，分子动力学模拟揭示MEZ亚基在开放和闭合状态之间交替，并且MEZ可以以多种构象稳定地结合其NAD（P）+辅因子，包括无活性的紧凑NAD+形式。MEZ的结构和来自其动力学的见解可以一起用于设计靶向Mtb的MEZ抑制剂。
Tuberculosis (TB) is the most lethal bacterial infectious disease worldwide. It is notoriously difficult to treat, requiring a cocktail of antibiotics administered over many months. The dense, waxy outer membrane of the TB-causing agent, Mycobacterium tuberculosis (Mtb), acts as a formidable barrier against uptake of antibiotics. Subsequently, enzymes involved in maintaining the integrity of the Mtb cell wall are promising drug targets. Recently, we demonstrated that Mtb lacking malic enzyme (MEZ) has altered cell wall lipid composition and attenuated uptake by macrophages. These results suggest that MEZ provides the required reducing power for lipid biosynthesis. Here, we present the X-ray crystal structure of MEZ to 3.6 Å resolution and compare it with known structures of prokaryotic and eukaryotic malic enzymes. We use biochemical assays to determine its oligomeric state and to evaluate the effects of pH and allosteric regulators on its kinetics and thermal stability. To assess the interactions between MEZ and its substrate malate and cofactors, Mn2+ and NAD(P)+, we ran a series of molecular dynamics (MD) simulations. First, the MD analysis corroborates our empirical observations that MEZ is unusually disordered, which persists even with the addition of substrate and cofactors. Second, the MD simulations reveal that MEZ subunits alternate between open and closed states and that MEZ can stably bind its NAD(P)+ cofactor in multiple conformations, including an inactive, compact NAD+ form. Together the structure of MEZ and insights from its dynamics can be harnessed to inform the design of MEZ inhibitors that target Mtb.