Structural Analysis of Biological Membrane Proteins

生物膜蛋白的结构分析

• 批准号: 9343593
• 负责人: di s xia
• 金额: $ 79.33万
• 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/9343593
• 关键词: 3-Dimensional; ABCB1 gene; ABCG2 gene; ATP Hydrolysis; ATP-Binding Cassette Transporters; Accounting; Achievement; Aconitate Hydratase; Active Biological Transport; Affinity; Antineoplastic Agents; Area; Behavior; Binding; Biochemical; Bioenergetics; Biological; Biological Models; Cattle; Cell physiology; Cell surface; Citric Acid Cycle; Clinic; Complex; Computer Simulation; Coupled; Coupling; Cytochrome bc1 Complex; Data Reporting; Drug resistance; Electron Transport; Electrons; Elements; Energy Metabolism; Enzymes; Family; Genetic; Glycogen Branching Enzyme; Goals; Heme; Human; Hydroquinones; Infection; Integral Membrane Protein; Iron-Sulfur Proteins; Knowledge; Lead; Link; Literature; Malate Dehydrogenase; 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; Pharmaceutical Preparations; Physiological; Plant Roots; Precipitation; Preparation; Protein Conformation; Proteins; Proton Pump; Protons; Quality Control; Reaction; Regulation; Research; Resolution; Respiratory Chain; Shapes; Site; Staging; Structural Models; Structure; Structure-Activity Relationship; Surface; Techniques; Testing; Therapeutic; Time; Work; X-Ray Crystallography; base; cancer therapy; high reward; high risk; human genome sequencing; inhibitor/antagonist; insight; interest; microbial; 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; ubiquinol; virtual; yeast genome

Multidrug resistance (MDR) is a long-standing clinic challenge in cancer therapies and in treatment of microbial infections. A major cause of MDR is the 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 elucidation of the structure at atomic resolution 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 determined structures of a stabilized mouse P-gp in various forms by X-ray crystallography, which afford us the ability to analyze structural basis of P-gp function. More importantly, this success offered us an opportunity to investigate the differences in solution behavior between human and mouse P-gp, which potentially may lead to the structure solution of human 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) 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.

多药耐药性（MDR）是癌症疗法和微生物感染治疗的长期临床挑战。 MDR的主要原因是外排ABC转运蛋白的过度表达，例如细胞表面上的P-糖蛋白（P-GP）。在癌症治疗期间停止P-gp的努力尚未成功。我的实验室一直在阐明P-gp原子分辨率的结构很长时间以来，试图从结构的角度揭示P-gp功能的机制。最近，我们通过X射线晶体学以各种形式成功地确定了稳定的小鼠P-gp的结构，这使我们能够分析P-gp功能的结构基础。更重要的是，这一成功为我们提供了一个机会，可以研究人与小鼠P-gp之间解决方案行为的差异，这可能会导致人类P-gp的结构解决方案。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。 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。