结核分支杆菌新药靶RpsA与POA作用阻断反式翻译的结构基础

Tuberculosis (TB) represents one of the world's greatest sources of mortality and morbidity. Mycobacterium tuberculosis （Mtb）, the pathogen responsible for TB, uses diverse strategies to survive in a variety of host lesions and to evade immune surveillance. The emergence of multi-drug-resistant strains of M. tuberculosis makes the discovery of new molecular scaffolds a priority, and the identification of new targets in M. tuberculosis that might be inhibited to effectively kill the existing strains is now a global pursuit. A new study, published in Science recently, suggests that pyrazinoic acid (POA), the active moiety of the first-line tuberculosis drug named Pyrazinamide (PZA), binds to the C domain of ribosomal protein S1 (RpsA) and inhibits trans-translation, which is essential for cell survival under stressful conditions. This finding identifies a potential promising target for new drugs and opens up a way for designing a new generation of antibiotics. However, the structure of Mtb. RpsA has not yet been determined, and it is still elusive how POA inhibit the interaction between RpsA and RNA. Besides it is reported that the C terminus of RpsA (RpsA_C) is involved in trans-translation by specifically binding to transfer-messenger RNA. Therefore, on the basis of our previous works, this project aims to determine the solution structures and the dynamics of RpsA_C, complexs POA-RpsA_C and RNA_14-RpsA_C by using multi-dimensional heteronuclear NMR spectroscopy. The interactions of RpsA with either POA or three kinds of RNAs will also be investigated respectively. We will focus on their binding sites and binding affnities, and we also analyze and compare the conformations of free-form with complex-form of RpsA_C. The study will provide the structure basis for understanding the interaction between RpsA and POA for inhibiting trans-translation, and it will provide the theoretical data for the development of tuberculosis drugs and other new antibiotics.

结核病（TB）是全球死亡率和发病率最高的传染性疾病之一，结核分枝杆菌(Mtb)是引起人类结核病的主要病原。随着全球耐多药结核菌株的出现和蔓延，迫切需要寻找新药靶及研发新药。2011年Science报道抗结核一线前药吡嗪酰胺(PZA)的体内水解物吡嗪酸(POA)与Mtb核糖体蛋白RpsA的C端（RpsA_C）结合阻断Mtb的反式翻译致使细胞中毒。这一发现使RpsA成为引人瞩目的治疗TB新药靶。然而，POA抑制RpsA蛋白与RNA作用的分子机制尚未清楚。本项目拟在前期工作的基础上，采用异核多维核磁共振技术，测定RpsA_C及两种复合物RpsA_C-POA和RpsA_C-RNA_14的溶液结构和动力学特征，研究RpsA_C分别与POA和三种RNA的相互作用,比较RpsA_C自由态与复合态的结构和动力学特征，揭示RpsA与POA结合阻断反式翻译的结构基础，为细菌相关疾病的药物研发提供科学理论依据。

本项目采用了多种谱学方法揭示PZA 耐药性的原因以及POA与结核分枝杆菌核糖体蛋白RpsA作用阻断反式翻译的结构基础。主要取得两方面的进展：第一，获得了MtRpsA 蛋白的C-terminal 结构域(MtRpsACTD)、MtRpsACTD-POA 复合物以及两个与PZA 耐药性突变相关的蛋白MtRpsAA438 和MsRpsA 的C-terminal 结构域的晶体结构（PDB code：4NNG、4NNH、4NNI），发现POA 靶向MtRpsA 主要是通过氨基酸Lys303、Phe307、Phe310、Glu318 和Arg357 介导的氢键和疏水作用进行的，其中Phe307、Phe310和Arg357 是参与tmRNA 结合的保守氨基酸；MtRpsACTD 结合POA 后引起了S1-domain 的β2、β3 和β5 的构象产生较大的变化，进一步破坏了MtRpsA 与tmRNA 的相互作用，进而导致反式翻译过程的终止，而RpsA的C-terminus 的氨基酸缺失或突变可引起POA结合口袋的氨基酸的构象发生变化，导致了POA 结合能力的丧失，从而产生PZA 耐药性。第二，测定了MtRpsACTD_S1、的自由态的三维溶液结构和动力学特征（BMRB code： 26725,； PDB code： 5IE8）。 MtRpsACTD_S1的15N主链驰豫数据表明，L326-V333的NOE数值低于平均值0.73，它们在ps-ns时间尺度内有较大幅度的、比较不受限的运动，这与其无规卷曲的性质相符，而H322具有最大NOE值（0.94）；四个较大的构象交换速率Rex值分布于残基V321 (19.93 s-1)、H322 (13.6 s-1)、V331 (21.47 s-1)、D335 (23.25 s-1)，该结果与低频谱密度函数J(ω0)一致；S2平均值为0.93，说明其整体结构比较刚性，Q295-K300、E314-E318、R329-H330和D343-V346区域的S2数值分布于0.7-0.9之间，这些区域在ps-ns时间尺度内有本体的结构柔性。H322的亲水侧链位于β桶的外侧，并且邻近POA/RNA的结合表面，它的这种具有构象交换特征的动力学性质可能可以介导不同的配体分子POA或RNA结合于MtRpsACTD_S1。