Structural Analysis of Biological Membrane Proteins

生物膜蛋白的结构分析

基本信息

批准号：
9153544
负责人：
di s xia
金额：
$ 75.42万
依托单位：
DIVISION OF BASIC SCIENCES - NCI
依托单位国家：
美国
项目类别：
财政年份：
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/9153544
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 Solutions 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 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

项目摘要

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）是癌症治疗和微生物感染治疗中长期存在的临床挑战。 MDR 的一个主要原因是细胞表面外排 ABC 转运蛋白（例如 P-糖蛋白 (P-gp)）的过度表达。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致力于P-gp原子分辨率的结构阐明，试图从结构角度揭示P-gp的功能机制。最近，我们通过X射线晶体学成功确定了多种形式的稳定小鼠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研究人类和小鼠 P-gp 解决方案行为差异的机会，这可能会导致人类 P-gp 的结构解决。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。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。