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题名: 雌激素受体α介导信号通路关键环节分子机制的计算研究
作者: 曹慧明1
学位类别: 博士
答辩日期: 2017-05
授予单位: 中国科学院生态环境研究中心
授予地点: 北京
导师: 张爱茜
关键词: 雌激素受体α信号通路, 分子动力学模拟 ; 配体结合 ; ERα-mediated signaling pathway, Molecular dynamics simulations,Ligand binding, Cofactor recruitment, ERE recognition ; 辅因招募 ; 受体响应元件识别
其他题名: Computational Study on Molecular Mechanism of Selected Key Steps in ERα-mediated Signaling Pathway
学位专业: 环境科学
中文摘要: 雌激素受体 α(ERα)作为核受体(NR)超家族的一员,参与着多种生理过 程的调节,与人体健康和疾病之间有着密不可分的联系。由其介导的信号通路是 环境污染物内分泌干扰效应的一个重要途径。在乳腺癌中,ERα因其显著的超活 化和过表达,导致癌细胞的增殖与发展,被作为重要的抗癌药物作用靶点受到广 泛关注。未激活时,雌激素受体结合伴侣蛋白呈现休眠状态。随着内源性激素进 入其配体结合域(LBD),使之从伴侣蛋白中解离,迅速形成二聚体。同时,其 LBD中的螺旋 H12构象发生改变,暴露表面的 AF-2位点利于随后的共激活辅因 子的招募。而后雌激素受体通过其 DNA结合域(DBD)识别特定的基因片段雌 激素响应元件(ERE),进而激活靶基因。遗憾的是,现阶段污染物经 ERα介导 内分泌干扰效应机制的计算研究多集中于配体结合这一步,忽略了污染物结合所 致受体构象调整所致辅因子招募等环节的功能异常对于最终健康结局的干扰,低 估了污染-受体作用微环境条件的差异所致受体关键残基质子化状态变化对整个 转录激活过程的影响。基于此,本论文综合运用量子化学、分子对接、分子动力 学和相关性网络分析等多种计算与分析技术,建立发展了能够对污染物结合所致 ERα变构引发的后续生物学关键环节的评价方法,针对配体结合、共激活因子招 募以及特定 DNA序列识别等 ERα介导信号通路中关键环节的分子机制进行模拟 研究,以期揭示与人体健康密切相关的环境污染物内分泌干扰效应的分子机制, 为保障环境安全和人体健康提供理论基础和方法支持。 论文首先针对一类典型的小体积环境拟/抗雌激素 BPA及其类似物 BPAF和 BPC开展其 ERα介导信号通路关键环节分子机制的计算研究。分析实验报道的 该类物质与 ERα的共结晶结构,发现拟雌激素 BPA和抗雌激素 BPC拥有截然不 同的 ERα结合模式,而 BPAF则是在受体二聚体的 2个单体中分别以这两种结 合姿态存在。因此,本研究已知的复合物晶体结构为基础,通过量子化学方法计 算环境污染物周围氨基酸对复合物稳定性的的能量贡献,发现 His524的能量贡 献减弱是 BPA和 BPAF与 ERα激动构型结合亲和力降低的主导原因。而 Thr347 作为 BPC识别中能量贡献与其他激动剂分子差异最明显的氨基酸,被推测在识 别 BPC等小尺寸拮抗剂中起到不可忽视的作用。为证实这一科学猜测,随后构 建了一系列 Thr347不同侧链二面角的复合物模型,进行了长时间的分子动力学 模拟,结合 MM-GBSA结合自由能计算证实与激动构型 ERα错配的 BPC能通过 诱导契合使 Thr347的侧链二面角发生反转,从而与其酚羟基形成强的氢键作用。 对 PDB中已有 ERα复合物晶体结构进行统计亦证实 Thr347侧链二面角数值在 结合激动剂和拮抗剂时存在显著差异。配体-蛋白-辅因子三元复合物分子动力学 模拟证实 BPC与典型拮抗剂 OHT均能通过结合 ERα间接地改变其 AF-2表面的 构象,进而干扰随后的共激活辅因子招募,最终导致三元复合物的整体稳定性下 降,结合亲和力减弱。同时,内在网络分析明确指出 Thr347在 BPC与 ERα相 互识别中采取的构象会削弱了 H3和 H12以及辅因子之间的相关性连接,不利于 共激活辅因子招募。基于计算获得的 ERα小体积拮抗剂干扰转录活化过程的结 构基础,本研究在 BPC结构基础上设计了与 ERα亲和力更强的抗雌激素,为乳 腺癌药物的设计提供了新思路。 ERα与小分子、伴侣蛋白、辅因子乃至 ERE作用时,若其关键残基质子化 状态发生变化,必然会影响这种分子间的相互识别和结合模式及强度。在 ERα 配体结合口袋和 DNA结合域中就存在着在生理 pH条件下质子化状态会发生变 化的残基,其在与具有氢键给受体或者可解离基团分子相互作用时,会因所处微 环境的不同采取不同的质子化状态而导致不同的分子间作用模式。本研究首先以 酸性极高的全氟烷酸类化合物(PFAAs)作为在生理状态以负离子而非分子形态 存在的 ERα配体为研究对象,解析 His524质子化状态对 PFAAs的 ERα结合姿 态和受体亲和性的影响机制。PFAAs不同于已知的 ERα激动剂/拮抗剂,其结构 中既没有苯环,也没有羟基基团,但仍表现出一定的 ERα结合亲和力以及拟雌 激素效应。针对该类具有直链结构的离子型有机化合物,我们通过基于多个蛋白 结构的集合对接的模拟策略,预测了这类生理条件下完全电离的污染物与 ERα 的结合能力,发现中等碳链长度的 PFOA、PFOS、PFDA和 PFNA与 ERα亲和 力排在前列,这与实验检测的转录活化能力结果相符合,暗示结合能力越强的化 合物其诱导产生的雌激素效应也越强。进一步分析 PFOA和 PFOS的结合模式发 现,位于结合口袋内的关键残基 His524的质子化状态会显著影响其结合姿态。 设置不同的 His524质子化形态,PFOA和 PFOS中极性羧酸或者磺酸基团会出现 朝向口袋底部 Arg394或者口袋入口 His524的两种不同结合指向。这驱使我们对 不同的结合模式进行分子动力学模拟和结合自由能计算 ,评价 PFOA/PFOS-ERα 复合物在水环境中的动态结合过程。结果显示 PFOA和 PFOS羧酸或者磺酸基团 朝向口袋起始处带正电的 His524的结合模式更稳定,而极性端朝向口袋底部的 Arg394的结合模式则降低了激动构型复合物的稳定性。随后的 MM-PBSA结合 自由能计算指出前一姿态结合亲和力强于后者是因为完全质子化的 His524能通 过其带正电荷的咪唑侧链与 PFOA和 PFOS的极性端形成稳定的氢键相互作用, 提供有利的静电相互作用贡献,从而稳定配体在口袋内的构象。而未质子化的 His524则不能作为好的氢键供体,PFOA和 PFOS因此翻转其姿态,调整其极性 基团指向,进而与底部的 Arg394形成稳定性较弱的氢键。以上结果证实体内酸 碱和静电相互作用微环境影响关键识别位点 His524的质子化形态,调控 PFOA 和 PFOS在 ERα的配体结合口袋内的结合姿态,从而影响其转录激活的强度。 实验报道 ERα的 DBD识别保守 DNA片段 cERE的能力受 His196和 Glu203 质子化状态的影响,但其分子机制和生物学意义尚不完全清晰。本研究依次构建 了不同质子化状态的 DBD-cERE复合物,对其构象稳定性进行了分子动力学模 拟评价。结构分析和结合自由能计算证实中性且氢原子位于 ε位的 His196和带 负电的 Glu203存在于高 pH环境,而带正电的 His196和中性的 Glu203存在于低 pH环境下。DBD与 cERE表面接触的关键残基的能量贡献暗示完全质子化且带 正电的 His196和 Glu203增强了其与碱基 DC34和 DA3的特异与非特异识别, 同时可间接控制 Arg211对于碱基 DT31和 DG32的识别。进一步构建代表性的 乳腺癌雌激素调节基因 pS2识别 ERα的特异性序列,通过分子动力学探查了pH 对 DBD与 pS2相互识别调控机制。结果指出 pS2在低 pH下不仅具有与cERE 相同的对于 DBD上 His196、Glu203和 Arg211识别增强位点,还因其特定序列 的改变增强了对于 Tyr197和 Gln238的识别能力。以上研究对于深度了解依赖 ERα转录活化的乳腺癌细胞增殖有所裨益。
英文摘要: The estrogen receptors α (ERα), as a member of the nuclear receptors superfamily, mediates various physiological processes and is strongly associated with the health issues and diseases. The ERα-mediated signaling pathway plays an important role in endocrine disrupting effect of environmental pollutants. The notable hyperactivation and overexpression of ERα have been detected in breast cancer cell, which result in the cell proliferation and disease development. Thus, ERα has received widespread attention as a drug target for breast cancer. Inactive ERα binds with its chaperone and keeps dormant. Once endogenous hormones bind into the ligand-binding domain (LBD) of ERα, the receptor dissociates from its complex with the chaperone proteins, followed by a dimerization process. The helix 12 of the LBD undergoes the conformational rearrangement for subsequent cofactor recruitment. Eventually, DNA-binding domain (DBD) of ERα interacts directly with a specific DNA sequence motif, estrogen response element (ERE), for transcriptional activation. Unfortunately, the present study on the mechanism of ERα-mediated endocrine disrupting effects mainly focuses on the ligand binding step and thus neglect the role of the other processes like cofactor recruitment, which can be accelerated or hindered by the conformational rearrangement of ERα, and underestimates the effects of change in chemical microenvironment on the protonation state of key residues during the whole transcriptional activation process. In order to tackle the problem, we explored an integrated computational method coupling various techniques, such as quantum chemistry calculation, molecular docking, molecular dynamics (MD) simulation and correlation network analysis, to evaluate the key steps of ERα-mediated signaling pathway, like ligand binding, cofactor recruitment and recognition of special DNA sequences as well. The achievements obtained from the study help to reveal the molecular mechanism of endocrine disrupting effects for typical environmental pollutants and provides the theoretical basis and methodology support to ensure environmental safety and human health. The dissertation first selected a kind of environmental estrogens with small molecular volume, bisphenol A (BPA) and its analogues, to elucidate the mechanisms as how the chemicals interfere with the key steps of ERα-mediated signaling pathway by using the established computational method. A structural scrutiny of crystal structures of ERα cocrystalized with ligands indicated that the binding mode of BPA is different from that of bisphenol C (BPC), while bisphenol AF (BPAF) adopts above two binding modes in the respective monomer of ERα dimer. Based the obtained crystal structures, we evaluated the energy contributions of the individual residues surrounding the ligands using the quantum chemistry calculation. His524 was identified as a main reason for reducing the binding affinity of BPA and BPAF to ERα, whereas with regard to antagonist with small molecular volume such as BPC, Thr347 was identified as a key residue for molecular recognition. In order to testify the above working hypothesis, we built a series of the complex models with different dihedral angle of Thr347 side chain for long time molecular dynamics simulations. The MM-GBSA calculations demonstrated that the binding of BPC into an agonist conformation of ERα could induce a flip of side chain dihedral angle of Thr347 for forming the hydrogen bonding interaction with phenol group of BPC. A systematic investigation of ERα crystal structures indicated there is significant difference in the side chain dihedral angle of Thr347 between agonist and antagonist conformations. Furthermore, the ternary complexes of ligands-ERα-cofactor also underwent MD simulations. The obtained results implied that the binding mode of BPC and typical antagonist OHT could indirectly affects the conformation of AF-2 and thus interferes with the recruitment of cofactor, thereby resulting in a stability decrease for the ternary complex and reducing the binding affinity of cofactor. Meanwhile, the network analysis of complex indicated that the binding of BPC could weaken the correlation connections between cofactor and H3 or H12. In addition, based on the structural basis for small volume molecules interfering with the transcriptional activation of ERα, we designed BPC analogues with strong affinity to ERα, which is rather helpful to the drug development for breast cancer. When ERα interacting with the small molecules, chaperonin, cofactor and ERE, the effect of the protonation states of key residues on molecular recognition and binding affinity cannot be neglected. In fact, the protonation state of selected ERα residues in LBD and DBD, such as His196, Glu203 and His524, are sensitive to the pH change in the physiological pH condition. Therefore, molecular recognition of ERα involving these residues is largely regulated by their pH- and electrostatic field- dependent protonation. In this regard, to elaborate the effect of protonation state of His524 on the binding modes and affinity of ligands with ERα, perfluoroalkyl acids (PFAAs) were selected as strong acidic chemicals with anions as their dominant speciation in the physiological condition. As weak ERα ligands with estrogenic effects, PFAAs are different from the other ligands of ERα since they present no common structural features of typical ERα agonists possessing phenol fragment in molecular structure. We employed the ensemble docking strategy to predict the ERα binding affinity of PFAAs. The obtained results indicated that perfluorooctanoic acid (PFOA), perfluorooctane sulfonate(PFOS),perfluorodecanoic acid(PFDA)and perfluorononanoic acid (PFNA) have top rank of binding affinity in the docking predictions, which is in line with their reported estrogenic effects in experimental measurement. Thus, it suggested the higher the binding affinities are, the stronger estrogenic effects become. Moreover, the protonation state of the key residue His524 were found to dramatically affect the prevailing binding poses of PFOA and PFOS in the active pocket of ERα. In docking simulations, the carboxyl and sulfonic acid groups of the ligands demonstrated two orientations in ERα, one pointing toward the pocket entrance, another extending to the cavity bottom. Further insight into the different binding modes of PFOA/PFOS-ERα complexes in aqueous solution has been gotten via molecular dynamics simulation and binding free energy calculation. The structural stability analysis suggested that the polar heads of the ligands preferred to the orientation providing hydrogen bonding with the positively charged His524, whereas the opposite orientation was towards Arg394 and led to reduced stability of these complexes. Subsequent MM-PBSA calculations verified that the strong intermolecular interaction established by hydrogen bonding between the positively charged imidazole moiety of His524 (+1 charged, both δ- and ε-nitrogen atoms protonated) and the negatively charged polar heads of the ligands provided additional stability to the complexes, which the weak hydrogen bonds of the polar head with Arg394 could not afford. However, the neutral imidazole ring of His524 with only either δ- or ε-nitrogen protonated is not a good hydrogen bond donor, thus cannot form hydrogen bonds with the ligands, and finally results in a pose inversion of the ligands in the pocket to point their polar groups toward Arg394 to enable the formation of unstable hydrogen bonds with the residue. The above results indicate that the cell microenvironment like pH can mediate the ligand recognition of ERα by affecting the protonation state of His524, thereby change the binding poses of PFOA and PFOS in the active pocket and subsequent transcriptional activation process. Recent experimental studies reported that the DBD-ERE molecular recognition for the conserved DNA sequence of ERα also relies on the protonation state of His196 and Glu203 somehow. However, its underlying mechanism remains unclear. Therefore, Molecular dynamics simulations were performed in the study to explore the conformational stability of the DBD-ERE complexes with two residues of different protonation states. The structural analysis and binding free energy calculations identified the electrically neutral Glu203 and the positively charged His196 with both δ- and ε-nitrogen atoms protonated as the typical state of two residues at low pH, while the negatively charged Glu203 and the electrically neutral His196 with the protonation of the ε-nitrogen on its imidazole were adopted at high pH. The energy contribution of individual residues to the DBD-ERE complexes implied that acidic condition prompt the molecular recognition since both specific and nonspecific base recognition for DC34 and DA3 could be enhanced by positively charged His196 and electrically neutral Glu203. Besides, the specific protonation state of the two residues at low pH helped the key residue Arg211 to successfully recognize the DT31-attached phosphate backbone and the base DG32. Further computational study on the ERα-DBD recognition by pS2/TFF1 gene, a typical ERα-responsive gene in breast cancer cells, revealed that the acidic condition not only enhanced the DNA recognition of His196, Glu203 and Arg211, but also improved the recognition of Tyr197 and Gln238 due to the change in special base sequence. These findings provide a better understanding of the molecular basis of the cell proliferation for breast cancer, largely depending on the transcriptional activation of ERα.
内容类型: 学位论文
URI标识: http://ir.rcees.ac.cn/handle/311016/38602
Appears in Collections:环境化学与生态毒理学国家重点实验室_学位论文

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作者单位: 1.中国科学院生态环境研究中心

Recommended Citation:
曹慧明. 雌激素受体α介导信号通路关键环节分子机制的计算研究[D]. 北京. 中国科学院生态环境研究中心. 2017.
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