Structural Basis of Biological Membrane Protein Functions and Drug Resistance

生物膜蛋白功能和耐药性的结构基础

• 批准号: 10014348
• 负责人: di s xia
• 金额: $ 111.95万
• 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/10014348
• 关键词: 3-Dimensional; ATP Hydrolysis; ATP phosphohydrolase; ATP-Binding Cassette Transporters; Aconitate Hydratase; Active Biological Transport; Address; Affinity; Anabolism; Area; Behavior; Binding; Biochemical; Bioenergetics; Biological; Bos taurus structural-GP protein; Cattle; Cell physiology; Cell surface; Citric Acid Cycle; Clinic; Complex; Computer Simulation; Couples; Coupling; Cytochrome bc1 Complex; Cytochrome c1; Data Reporting; Development; Drug resistance; Electron Transport; Electron Transport Complex III; Electrons; Elements; Energy Metabolism; Family; Glycogen Branching Enzyme; Goals; Heme; Human; Hydroquinones; Individual; Infection; Intervention; Iron-Sulfur Proteins; Knowledge; Lead; Length; Link; Literature; Lobe; Malate Dehydrogenase; Malignant Neoplasms; Membrane; Membrane Proteins; Membrane Transport Proteins; Methods; Methylation; Mitochondria; Molecular; Molecular Conformation; Motion; Movement; Multi-Drug Resistance; Mus; Oxidoreductase; Oxygen; P-Glycoproteins; Pharmaceutical Preparations; Protein Conformation; Protein Subunits; Proteins; Proton Pump; Protons; Regulation; Research; Research Design; Resistance; Resolution; Respiratory Chain; Rhodamine 123; Series; Shapes; Site; Structure; Structure-Activity Relationship; Substrate Interaction; Succinates; Surface; Testing; Therapeutic; Therapeutic Agents; Time; Transmembrane Domain; Work; base; calcein AM; cancer therapy; design; experimental study; flexibility; inhibitor/antagonist; interest; loss of function; microbial; mutant; novel; oxidation; pathogenic microbe; photosynthetic bacteria; programs; protein function; receptor; screening; small molecule; small molecule libraries; success; therapeutic protein; ubiquinol; virtual

Multidrug resistance (MDR) is a long-standing clinic challenge in cancer therapies and in treatment of microbial infections; it is defined by a simultaneous resistance or cross resistance to various unrelated therapeutic agents by cancers or microbial pathogens. One mechanism of MDR is the over expression of efflux ABC transporters such as human P-glycoproteins (hP-gp) on cell surface. The prospect of reversing the function of hP-gp im order to overcome MDR in cancer therapy has driven development of P-gp specific inhibitors. However, such efforts have so far been unsuccessful, despite extensive studies designed to elucidate the underlying mechanism of function of these P-gp inhibitors. One issue is clearly related to the lack of detailed structural knowledge of P-gp and the solution is to obtain the structure of hP-gp in complex with these inhibitors such that detailed interactions can be revealed. As a first step, we must obtain the structure(s) of hP-gp in its native form and in various conformations. My lab has been working on the elucidation of the structure at atomic resolution of hP-gp for a long time in our attempts to uncover the mechanism of P-gp function from a structural perspective. Some of the questions we would like to address are (1) understanding the structural basis of P-gp substrate polyspecificity, (2) the coupling of ATP hydrolysis to the substrate translocation, and (3) the mechanism of P-gp inhibition. For many years, the structure determination of P-gp by the crystallographic method has been hampered by its intrinsic flexibility that is facilitated by a 75-residue linker connecting the two halves of P-gp. We shortened the linker to facilitate the structure determination of mP-gp, which were subsequently used for successful structure determination of many other mP-gp structures. These structures lead to some very interesting findings outlined belew. (1) Despite dramatic reduction in rhodamine 123 and calcein-AM transport, the linker-shorteded mutant P-gp possesses a basal ATPase activity but has lost the drug-stimulated ATPase activity. (2) The linker-shortened mutant is structurally intact and surprisingly still has the same inward-facing conformation as that observed in the full-length P-gp, which suggests that the loss of function of the linker-shortened mutant is due to the loss of flexibility of the protein. (3) In the absence of substrate, P-gp only binds ATP asymmetrically in the NBD1, which is supported by our protective methylation experiment. (4) Analyses of a series structures of wild-type, linker mutant, and a methylated P-gp showed individual transmembrane-domain helices of P-gp undergoing significant movements, which, importantly, correlates strongly with the opening-and-closing movement of the two lobes of P-gp. Thus, the opening-and-closing motion of the two halves of P-gp alters the surface topology within its drug-binding pocket, providing a mechanistic explanation for the polyspecificity of P-gp in substrate interactions. This work afford us the ability to analyze the 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, as we hope, may lead to the structure solution of hP-gp. My lab has been studying the structure and funciton of the cytochrome bc1 complex from bovine mitochondria (Mtbc1, also known as Complex III of the respiratory chain) and the photosynthetic bacterium R. sphaeroides (Rsbc1) in various forms. Based on our structural and functional studies, we have proposed an hypothesis termed the surface-affinity modulated iron-sulfur protein (ISP) conformation switch to address the mechanism for the bifurcated electron transfer (ET) at the quinol oxidation (QP) site of the cytochrome bc1 complex. At the center of this hypothesis is the regulation of the distance between heme c1 of the cytochrome c1 subunit and the 2Fe2S cluster of the iron-sulfur protein subunit. The distance is too long to permit ET between the two sites when ISP-ED is in the fixed conformation; ET is only permissible 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. More recently, we have provided further experimental evidence to support this hypothesis by structure determinations of various Rsbc1 structures in complex with different inhibitors, which showed the switching of the conformation of iron-sulfur protein in the presence of different inhibitors. Over a decade of intensive 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 cyt bc1 by components of the TCA cycle such as malate dehydrogenase (MDH), aconitase (ACON) and succinate-ubiquinol dehydrogenase (Complex II) and by small molecules such as molecular oxygen. These studies are ongoing.

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；它的定义是癌症或微生物病原体对各种不相关的治疗剂同时产生耐药性或交叉耐药性。 MDR 的机制之一是细胞表面外排 ABC 转运蛋白的过度表达，例如人 P-糖蛋白 (hP-gp)。在癌症治疗中逆转 hP-gp 功能以克服 MDR 的前景推动了 P-gp 特异性抑制剂的开发。然而，尽管进行了广泛的研究来阐明这些 P-gp 抑制剂的潜在功能机制，但迄今为止，此类努力尚未成功。其中一个问题显然与缺乏 P-gp 的详细结构知识有关，解决方案是获得与这些抑制剂复合的 hP-gp 的结构，以便揭示详细的相互作用。第一步，我们必须获得 hP-gp 的天然形式和各种构象的结构。我的实验室长期致力于hP-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。我们想要解决的一些问题是（1）了解 P-gp 底物多特异性的结构基础，（2）ATP 水解与底物易位的耦合，以及（3）P-gp 抑制的机制。多年来，通过晶体学方法测定 P-gp 的结构一直受到其固有灵活性的阻碍，而连接 P-gp 两半的 75 个残基连接体促进了这种灵活性。我们缩短了连接子以促进 mP-gp 的结构测定，随后将其用于许多其他 mP-gp 结构的成功结构测定。这些结构导致了下面概述的一些非常有趣的发现。 (1) 尽管罗丹明 123 和钙黄绿素-AM 转运显着减少，但接头短路的突变体 P-gp 具有基础 ATP 酶活性，但失去了药物刺激的 ATP 酶活性。 (2) 连接子缩短的突变体在结构上是完整的，并且令人惊讶地仍然具有与全长 P-gp 中观察到的相同的向内构象，这表明连接子缩短的突变体的功能丧失是由于蛋白质失去弹性。 (3)在没有底物的情况下，P-gp仅在NBD1中不对称地结合ATP，这得到了我们的保护性甲基化实验的支持。 (4) 对野生型、接头突变体和甲基化 P-gp 的一系列结构的分析表明，P-gp 的各个跨膜域螺旋经历了显着的运动，重要的是，这与打开和关闭运动密切相关P-gp 的两个叶。因此，P-gp 两半的打开和关闭运动改变了其药物结合口袋内的表面拓扑结构，为 P-gp 在底物相互作用中的多特异性提供了机制解释。这项工作使我们能够分析 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，正如我们所希望的那样，这可能会导致 hP-gp 的结构解决。我的实验室一直在研究来自牛线粒体的细胞色素 bc1 复合物（Mtbc1，也称为呼吸链复合物 III）和各种形式的光合细菌 R. sphaeroides (Rsbc1) 的结构和功能。基于我们的结构和功能研究，我们提出了一种称为表面亲和力调节铁硫蛋白（ISP）构象转换的假设，以解决铁硫蛋白对醌氧化（QP）位点的分叉电子转移（ET）机制。细胞色素 bc1 复合物。该假说的核心是细胞色素 c1 亚基的血红素 c1 与铁硫蛋白亚基的 2Fe2S 簇之间距离的调节。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 处于移动构象时才允许 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。最近，我们通过对与不同抑制剂复合的各种 Rsbc1 结构进行结构测定，提供了进一步的实验证据来支持这一假设，这表明铁硫蛋白在不同抑制剂存在下的构象发生了转变。十多年来的深入研究可以说已经解决了有关细胞色素 bc1 复合物的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奠定了基础，其中包括 TCA 成分对 cyt bc1 的调节循环，如苹果酸脱氢酶 (MDH)、乌头酸酶 (ACON) 和琥珀酸泛醇脱氢酶（复合物 II）以及小分子（如分子氧）。这些研究正在进行中。