Structural Analysis of Biological Membrane Proteins

生物膜蛋白的结构分析

• 批准号: 8937708
• 负责人: di s xia
• 金额: $ 85.81万
• 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/8937708
• 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; 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; yeast genome

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.

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）站点提供了支持该假设的实验证据，并首次在QP站点中鉴定出基喹醇（QH2）。所有这些成就都源于我们对更好衍射晶体的不懈追求。 RSBC1的结构解决方案实现了我们在建立模型系统来通过结合结构，遗传和生化技术来系统研究BC1复合物的目标之一。我们对牛BC1的结构研究使我们提出，QP位点上分叉ET的关键是对ISP-ED运动的控制，该运动调节了2FE2S簇和C1血红素之间的距离。当ISP-ED处于固定构象中时，距离太长允许两个位点之间的ET。 ET仅在移动构象中ISP-ED时才有可能。我们假设通过调节结合表面的形状，Cyt B亚基有效地控制了其对ISP-ED的亲和力，ISP的运动，从而从底物泛醇中的两个电子的方向进行了控制。来自文献中的报告和我们实验室的新实验的数据支持了这一假设。目前，我们专注于在没有抑制剂的情况下在实验中证明控制机制，这与生理条件更相关。可以说，在十年中，3D结构研究的大量研究已经解决了有关细胞色素BC1复合物的结构功能关系的大多数问题，为将这种重要复合物的知识纳入了阶段，以将BC1调节的较广泛的生物烯类综合体纳入较广泛的生物能源景观中。分子氧根据络合物的完整性，将BC1的电子转移活性提高了82％。氧对细胞色素BC1复合物的反应序列的影响在通过Q-Cycle机制的泛素醇的分叉氧化过程中血红素BL还原的速度。 TCA循环酶，苹果酸脱氢酶（MDH）和痤疮酶（ACON）之间的特定相互作用已通过共沉淀及其增强BC1活性的能力证明。这些相互作用的晶体研究正在进行中。自1970年代获得FDA的批准以来，顺铂化疗已成为广泛的癌症治疗方法的基石，它是当今癌症医学中最常用的化学疗法药物之一。像许多其他化学治疗剂一样，顺铂在其临床应用中面临着越来越多的抗药性问题。已经提出了许多机制来发展顺铂耐药性，包括药物的细胞摄取和外排的变化，增加药物排毒，抑制凋亡以及DNA修复的增加。尽管该领域进行了广泛的研究，但顺铂耐药性的分子机制仍然难以捉摸。假设蛋白TMEM205（以前称为MBC3205）被分泌的蛋白发现倡议推测为分泌的整体膜蛋白。该蛋白质由189个氨基酸残基组成，具有四个预测的跨膜螺旋（TMHS）。直到最近我们报道了其在癌细胞系中替铂耐药中的作用，对这种蛋白质及其细胞功能知之甚少。使用荧光标记的顺铂，结果表明，TMEM205的过表达降低了顺铂在癌细胞系中的积累。这种还原与细胞的顺铂抗性相关。 TMEM205被证明是一种位于细胞表面的膜蛋白，该膜在人体耐抗链铂颈癌和内部在反式高尔基网络附近的内部耐颈癌和肝癌细胞中高度表达。该蛋白的高表达水平在某些分泌组织中，例如肝脏，胰腺和肾上腺的组织，与TMEM205在顺铂抗性中的作用一致。为了实现TMEM205的结构溶液，我们过表达了该积分膜蛋白，确定了其寡聚状态并将其结晶。 TMEM205晶体衍射X射线至2 A分辨率。目前，我们正在努力解决晶体学期问题。多药耐药性（MDR）是癌症疗法的长期诊所挑战。 MDR与细胞表面上排出ABC转运蛋白（例如P-糖蛋白（P-GP））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。我的实验室长期以来一直在阐明P-gp的结构，以从结构的角度揭示P-gp功能的机制。最近，我们在酵母表达系统中成功表达了人类和小鼠P-gp。更重要的是，我们能够将小鼠P-gp和小鼠P-gp的晶体衍射为3.0 A分辨率。这一成功为我们提供了研究人和小鼠P-gp的溶液行为差异，并有可能提供小鼠P-gp结构的差异。我的实验室还参与了ABC转运蛋白的分子建模研究，该研究已成为获得对原子结构未知的蛋白质结构和功能见解的重要工具。多年来，我们为许多ABC转运蛋白（例如ABCB1，ABCG2，PDR5P等）构建了结构模型。这些模型可作为这些蛋白质进一步特征的指导。