Structural Analysis of Biological Membrane Proteins

生物膜蛋白的结构分析

• 批准号: 8763073
• 负责人: di s xia
• 金额: $ 71.84万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8763073
• 关键词: 3-Dimensional; ABCB1 gene; ABCG2 gene; ATP Hydrolysis; ATP-Binding Cassette Transporters; Accounting; Achievement; Aconitate Hydratase; Active Biological Transport; Adrenal Glands; Affinity; Amino Acids; Antineoplastic Agents; Area; Behavior; Binding; Biochemical; Bioenergetics; Biological; Biological Models; Cancer cell line; Cattle; Cell physiology; Cell surface; Cells; Cervix carcinoma; Cisplatin; Citric Acid Cycle; Clinic; Complex; Computer Simulation; Coupled; Coupling; Cytochrome bc1 Complex; DNA Repair; Data Reporting; Development; Drug Efflux; Drug Metabolic Detoxication; Drug resistance; Electron Transport; Electrons; Elements; Energy Metabolism; Enzymes; Family; Fluorescence; Fungal Genome; Genetic; Goals; Heme; Human; Human Genome; Hydroquinones; Inhibition of Apoptosis; Integral Membrane Protein; Iron-Sulfur Proteins; Knowledge; Label; Literature; Liver; Malate Dehydrogenase; Malignant Epithelial Cell; Malignant Neoplasms; Medicine; Membrane; Membrane Proteins; Membrane Transport Proteins; Methodology; Mitochondria; Modeling; Molecular; Molecular Conformation; Molecular Models; Movement; Multi-Drug Resistance; Mus; Open Reading Frames; Oxygen; P-Glycoproteins; PAWR protein; Pancreas; Pharmaceutical Preparations; Phase; Physiological; Plant Roots; Precipitation; Preparation; Protein Conformation; Proteins; Proton Pump; Protons; Quality Control; Reaction; Regulation; Reporting; Research; Resistance; Resolution; Respiratory Chain; Roentgen Rays; Role; Shapes; Site; Solutions; Staging; Structural Models; Structure; Structure-Activity Relationship; Surface; System; Techniques; Testing; Therapeutic; Time; Tissues; Work; X-Ray Crystallography; Yeasts; base; cancer therapy; chemotherapeutic agent; chemotherapy; clinical application; genome sequencing; hepatoma cell; high reward; high risk; inhibitor/antagonist; insight; interest; molecular modeling; overexpression; oxidation; photosynthetic bacteria; programs; protein expression; protein function; protein structure; receptor; research study; screening; small molecule libraries; structural biology; success; three dimensional structure; tool; trans-Golgi Network; ubiquinol; uptake; virtual

We have determined the structures of the cytochrome bc1 complex from bovine mitochondria (Mtbc1) and the photosynthetic bacterium R. sphaeroides (Rsbc1) in various forms, proposed an hypothesis for the mechanism of the surface-affinity modulated iron-sulfur protein (ISP) conformation switch to account for the bifurcated electron transfer (ET) at the quinol oxidation (QP) site, provided experimental evidence to support this hypothesis, and identified substrate ubiquinol (QH2) in the QP site for the first time. All these achievements were rooted in our relentless pursuit of better diffracting crystals. The structure solution of Rsbc1 accomplishes one of our goals in establishing a model system to systematically study the bc1 complex by combining structural, genetic, and biochemical techniques. Our structural studies of bovine bc1 led us to propose that the key to the bifurcated ET at the QP site is the control of the ISP-ED movement, which regulates the distance between the 2Fe2S cluster and c1 heme. The distance is too long to permit ET between the two sites when ISP-ED is in the fixed conformation; ET is only possible when ISP-ED is in the mobile conformation. We hypothesized that by modulating the shape of the binding surface, the cyt b subunit effectively controls its affinity for the ISP-ED, the movement of the ISP, and thereby the directions of the two electrons from the substrate ubiquinol. Data from reports in the literature and new experiments from our lab and from others support this hypothesis. Currently we are focusing on demonstrating the control mechanism in experiment in the absence of inhibitors, which is more relevant to physiological conditions. Over a decade of intensive post 3D-structure studies have arguably resolved most questions regarding the structure-function relationship of the cytochrome bc1 complex, setting the stage for integrating knowledge of this vital complex into a broader bioenergetics landscape that includes the regulation of bc1 by components of the TCA cycle and by molecular oxygen. Molecular oxygen enhances the electron transfer activity of bc1 by 82% depending on the intactness of the complex. The effect of oxygen on the reaction sequence of the cytochrome bc1 complex is at the step of heme bL reduction during the bifurcated oxidation of ubiquinol via the Q-cycle mechanism. Specific interactions between TCA cycle enzymes, malate dehydrogenase (MDH) and aconitase (ACON), have been demonstrated by co-precipitation and their ability to enhance bc1 activity. Crystallograpic studies of these interactions are underway. Since its approval by the FDA in the 1970s, cisplatin chemotherapy has become the cornerstone of a broad spectrum of cancer treatments and it is one of the most commonly used chemotherapy drugs in cancer medicine today. Like many other chemotherapeutic agents, cisplatin is facing a growing problem of resistance by cancers in its clinical application. A number of mechanisms have been proposed for the development of cisplatin resistance, including changes in cellular uptake and efflux of the drug, increased detoxification of the drug, inhibition of apoptosis, and increased DNA repair. Despite extensive research in the field, molecular mechanisms of cisplatin resistance remain elusive. The hypothetical protein TMEM205, formerly known as MBC3205, was speculated to be a secreted integral membrane protein by the Secreted Protein Discovery Initiative. This protein consists of 189 amino acid residues with four predicted trans-membrane helices (TMHs). Little was known about this protein and its cellular function until recently when we reported its role in cisplatin resistance in cancer cell lines. Using a fluorescence-labeled cisplatin, it was shown that overexpression of TMEM205 reduces accumulation of cisplatin in cancer cell lines; this reduction correlates with the cisplatin resistance of the cells. TMEM205 is shown to be a membrane protein localized to the cell surface andis highly expressed in both human cisplatin-resistant cervical carcinoma and hepatoma cells internally near the trans-golgi network. High expression levels of this protein are found in certain secretory tissues, such as those of the liver, pancreas, and adrenal glands, consistent with the postulated role of TMEM205 in cisplatin resistance. To achieve structural solution of TMEM205, we overexpressed this integral membrane protein, determined its oligomeric state, and crystallized it. The TMEM205 crystals diffracted X-rays to 2 A resolution. Currently, we are working to solve the crystallographic phase problem. Multidrug resistance (MDR) is a long-standing clinic challenge in cancer therapies. MDR is associated with over expression of efflux ABC transporters such as P-glycoproteins (P-gp) on cell surface. Efforts to stop P-gp during cancer treatment have not been successful. My lab has been working on elucidating the structure of P-gp for a long time in our attempts to uncover the mechanism of P-gp function from a structural perspective. Recently, we have successfully expressed both human and mouse P-gp in yeast expression systems. More importantly, we were able to crystallize mouse P-gp and crystals of mouse P-gp diffracted to 3.0 A resolution. This success provides us an opportunity to investigate the differences in solution behavior of human and mouse P-gp and potentially to provide a better structure of mouse P-gp. My lab also engages in molecular modeling studies of ABC transporters, which has become an important tool to gain structural and functional insights into proteins whose atomic structures are unknown. Over the years, we have constructed structural models for a number of ABC transporters such as ABCB1, ABCG2, Pdr5p, etc. These models are useful as guidance for further characterizations of these proteins.

我们确定了来自牛线粒体（Mtbc1）和光合细菌R. sphaeroides（Rsbc1）的多种形式的细胞色素bc1复合物的结构，提出了表面亲和力调节铁硫蛋白（ISP）构象机制的假设转而解释对苯二酚氧化（QP）位点的分叉电子转移（ET），提供了支持这一假设的实验证据，并确定了底物泛醇（QH2）首次出现在QP网站上。所有这些成就都源于我们对更好的衍射晶体的不懈追求。 Rsbc1的结构解决方案实现了我们建立模型系统的目标之一，通过结合结构、遗传和生化技术来系统地研究bc1复合物。我们对牛 bc1 的结构研究使我们提出 QP 位点分叉 ET 的关键是 ISP-ED 运动的控制，它调节 2Fe2S 簇和 c1 血红素之间的距离。当 ISP-ED 处于固定构象时，两个位点之间的距离太长，无法进行 ET；仅当 ISP-ED 处于移动构象时才可能进行 ET。我们假设通过调节结合表面的形状，cyt b 亚基有效地控制其对 ISP-ED 的亲和力、ISP 的运动，从而控制来自底物泛醇的两个电子的方向。来自文献报告以及我们实验室和其他实验室的新实验的数据支持了这一假设。目前我们的重点是在实验中展示在没有抑制剂的情况下的控制机制，这与生理条件更相关。十多年来深入的后 3D 结构研究可以说已经解决了有关细胞色素 bc1 复合物结构与功能关系的大多数问题，为将这一重要复合物的知识整合到更广泛的生物能学领域（包括成分对 bc1 的调节）奠定了基础TCA 循环和分子氧。根据复合物的完整性，分子氧可将 bc1 的电子转移活性提高 82%。氧对细胞色素 bc1 复合物反应顺序的影响是在泛醇通过 Q 循环机制的分叉氧化过程中血红素 bL 还原的步骤。 TCA 循环酶、苹果酸脱氢酶 (MDH) 和乌头酸酶 (ACON) 之间的特异性相互作用已通过共沉淀及其增强 bc1 活性的能力得到证明。这些相互作用的晶体学研究正在进行中。自 20 世纪 70 年代获得 FDA 批准以来，顺铂化疗已成为广泛癌症治疗的基石，也是当今癌症医学中最常用的化疗药物之一。与许多其他化疗药物一样，顺铂在临床应用中面临着日益严重的癌症耐药问题。已经提出了多种顺铂耐药性产生的机制，包括细胞摄取和药物流出的变化、药物解毒的增加、细胞凋亡的抑制和DNA修复的增加。尽管该领域进行了广泛的研究，但顺铂耐药的分子机制仍然难以捉摸。假设的蛋白质 TMEM205（以前称为 MBC3205）被分泌蛋白发现计划推测为分泌性整合膜蛋白。该蛋白质由 189 个氨基酸残基和四个预测的跨膜螺旋 (TMH) 组成。人们对这种蛋白质及其细胞功能知之甚少，直到最近我们报道了它在癌细胞系顺铂耐药性中的作用。使用荧光标记的顺铂，结果表明TMEM205的过度表达可减少癌细胞系中顺铂的积累；这种减少与细胞的顺铂耐药性相关。 TMEM205 被证明是一种定位于细胞表面的膜蛋白，并且在人顺铂耐药宫颈癌和跨高尔基体网络内部的肝癌细胞中高度表达。该蛋白在某些分泌组织中存在高表达，例如肝脏、胰腺和肾上腺，这与 TMEM205 在顺铂耐药性中的假设作用一致。为了获得 TMEM205 的结构解决方案，我们过表达了这种整合膜蛋白，确定了其寡聚状态，并将其结晶。 TMEM205 晶体将 X 射线衍射至 2 A 分辨率。目前，我们正在努力解决晶体相问题。多药耐药性（MDR）是癌症治疗中长期存在的临床挑战。 MDR 与细胞表面 P-糖蛋白 (P-gp) 等外排 ABC 转运蛋白的过度表达有关。在癌症治疗期间停止 P-gp 的努力尚未成功。我的实验室长期致力于阐明P-gp的结构，试图从结构角度揭示P-gp的功能机制。最近，我们在酵母表达系统中成功表达了人和小鼠的P-gp。更重要的是，我们能够结晶小鼠 P-gp 并将小鼠 P-gp 晶体衍射至 3.0 A 分辨率。这一成功为我们提供了研究人类和小鼠 P-gp 溶液行为差异的机会，并有可能提供更好的小鼠 P-gp 结构。我的实验室还从事 ABC 转运蛋白的分子建模研究，这已成为了解原子结构未知的蛋白质的结构和功能的重要工具。多年来，我们构建了许多 ABC 转运蛋白的结构模型，例如 ABCB1、ABCG2、Pdr5p 等。这些模型可作为进一步表征这些蛋白质的指导。