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

基本信息

批准号：
10925999
负责人：
di s xia
金额：
$ 273.06万
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/10925999
关键词：
3-Dimensional ATP Hydrolysis ATP phosphohydrolase ATP-Binding Cassette Transporters Aconitate Hydratase Active Biological Transport Address Affinity Agriculture Anabolism Antibiotics Area Automobile Driving Behavior Binding Biochemical Bioenergetics Biogenesis Biological Bos taurus Cattle Cell physiology Cell surface Cellular biology Chemotherapy-Oncologic Procedure Citric Acid Cycle Clinic Coenzyme Q10 Complex Core Assembly Couples Coupling Crystallography Cytochrome bc1 Complex Development Drug resistance Electron Transport Electron Transport Complex III Elements Energy Metabolism Family Focus Groups Glycoproteins Goals Human Hydroquinones Individual Infection Intervention Iron-Sulfur Proteins Knowledge Lead Length Link Lobe Malate Dehydrogenase Malignant Neoplasms Membrane Membrane Proteins Membrane Transport Proteins Methods Methylation Mitochondria Mitochondrial Membrane Protein Molecular Molecular Conformation Motion Movement Multi-Drug Resistance Mus Mutation Oxidoreductase Oxygen Pathway interactions Pharmaceutical Preparations Protein Conformation Protein Subunits Proteins Protons Regulation Research Research Design Resistance Resolution Respiratory Chain Rhodamine 123 Rhodobacter sphaeroides Series Site Structure Structure-Activity Relationship Substrate Interaction Succinates Surface Testing Therapeutic Therapeutic Agents Time Transmembrane Domain Ubiquinone Visualization Work Zebrafish calcein AM cancer therapy clinical application cytochrome c design experimental study flexibility in silico inhibitor interest knowledge integration loss of function microbial mutant novel overexpression oxidation pathogenic microbe photosynthetic bacteria programs protein function receptor respiratory small molecule small molecule libraries success therapeutic protein ubiquinol virtual screening

项目摘要

Cellular drug resistance is rendered by several mechanisms. From structural perspectives, my group focuses our study on the two most common ones: target site mutations and reduction of intracellular drug concentration. Target site mutation is one of the most common forms of drug resistance. My lab has been studying the structure and function of the cytochrome bc1 complex from bovine (Bos taurus) mitochondria (Btbc1, also known as Complex III of the cellular respiratory chain) and the photosynthetic bacterium R. sphaeroides (Rsbc1) in various forms. Complex III is a validated target for antibiotics targeting pathogenic microbes. Based on our structural and functional studies, we have proposed a hypothesis to address central mechanistic question of the Q-cycle mechanism for Complex III function. This hypothesis, termed the surface-affinity modulated iron-sulfur protein (ISP) conformation switch, addresses the mechanism for the bifurcated electron transfer (ET) at the quinol oxidation (QP) site of the cytochrome bc1 complex. We have provided further experimental evidence to support our 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 extensive 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. We have also been studying Complex III biogenesis by elucidating the structures of bcs1, a mitochondrial membrane protein that is critical in assisting insertion of the ISP subunit into core assembly of the Complex III. Reduction of intracellular drug concentration represents another important mechanism of drug resistance. Multidrug resistance (MDR) is a long-standing clinic challenge in cancer chemotherapies 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 the cell surface. The prospect of reversing the function of hP-gp in order to overcome MDR in cancer therapy has been driving 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 relating to various steps along its catalytic pathway and the solution is to obtain the structures 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 and mP-gp (mouse P-gp) for a long time, and more recently ZfP-gp (Zebra fish P-gp), in our attempts to uncover the mechanism of P-gp function, especially their ability to recognize structurally diverse compounds, 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 below. (1) Despite dramatic reduction in rhodamine 123 and calcein-AM transport, the linker-shortened 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 of 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 affords 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.

细胞耐药性是通过多种机制产生的。从结构角度来看，我的团队将研究重点放在两个最常见的方面：靶点突变和细胞内药物浓度降低。靶位点突变是最常见的耐药形式之一。我的实验室一直在研究来自牛 (Bos taurus) 线粒体的细胞色素 bc1 复合物（Btbc1，也称为细胞呼吸链复合物 III）和各种形式的光合细菌 R. sphaeroides (Rsbc1) 的结构和功能。复合物 III 是针对病原微生物的抗生素的经过验证的靶点。基于我们的结构和功能研究，我们提出了一个假设来解决复杂 III 功能的 Q 循环机制的核心机制问题。这一假说被称为表面亲和力调节铁硫蛋白 (ISP) 构象转换，解决了细胞色素 bc1 复合物的醌氧化 (QP) 位点的分叉电子转移 (ET) 机制。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我们的假设，这表明铁硫蛋白在不同抑制剂存在下的构象发生了转变。十多年来的广泛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。我们还通过阐明 bcs1 的结构来研究复合物 III 的生物发生，bcs1 是一种线粒体膜蛋白，对于协助 ISP 亚基插入复合物 III 的核心组装至关重要。细胞内药物浓度的降低是耐药性的另一个重要机制。多药耐药性（MDR）是癌症化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 的机制之一是外排 ABC 转运蛋白在细胞表面的过度表达，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 的前景一直在推动 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。一个问题显然与缺乏与催化途径各个步骤相关的 P-gp 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 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。我的实验室长期以来一直致力于以原子分辨率阐明 hP-gp 和 mP-gp（小鼠 P-gp）的结构，最近我们尝试了 ZfP-gp（斑马鱼 P-gp）的结构从结构角度揭示P-gp功能机制，特别是其识别结构不同化合物的能力。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，但接头缩短的突变体 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 的各个跨膜结构域螺旋经历了显着的运动，重要的是，这与打开和关闭密切相关。 P-gp 的两个叶的运动。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。