Structural Analysis of Biological Membrane Proteins

生物膜蛋白的结构分析

• 批准号: 8348974
• 负责人: di s xia
• 金额: $ 79.03万
• 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/8348974
• 关键词: 3-Dimensional; ABCB1 gene; ABCG2 gene; ATP Hydrolysis; ATP-Binding Cassette Transporters; Accounting; Achievement; Active Biological Transport; Adrenal Glands; Affect; Affinity; Amino Acids; Antineoplastic Agents; Area; Behavior; Belief; Binding; Biochemical; Biological; Biological Models; Cancer cell line; Cattle; Cell physiology; Cell surface; Cells; Cervix carcinoma; Cisplatin; 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; Family; Fluorescence; Fungal Genome; Genetic; Goals; Heme; Human; Human Genome; Hydrogen Bonding; Hydroquinones; Inhibition of Apoptosis; Integral Membrane Protein; Iron-Sulfur Proteins; Kinetics; Knowledge; Label; Literature; Liver; Malignant Epithelial Cell; Malignant Neoplasms; Measurement; Medicine; Membrane; Membrane Proteins; Membrane Transport Proteins; Methodology; Modeling; Molecular; Molecular Conformation; Molecular Models; Movement; Multi-Drug Resistance; Mus; Mutagenesis; Open Reading Frames; Oxidation-Reduction; P-Glycoproteins; PAWR protein; Pancreas; Pharmaceutical Preparations; Plant Roots; Preparation; Process; Property; Protein Binding; Protein Conformation; Proteins; Proton Pump; Protons; Quality Control; Reporting; Research; Resistance; Resolution; Respiratory Chain; Roentgen Rays; Role; Screening procedure; Shapes; Site; Solutions; Structural Models; Structure; Surface; System; Techniques; Testing; Therapeutic; Time; Tissues; Work; X-Ray Crystallography; Yeasts; base; cancer therapy; chemotherapeutic agent; chemotherapy; clinical application; conformational conversion; genome sequencing; hepatoma cell; high reward; high risk; inhibitor/antagonist; insight; interest; molecular modeling; mutant; overexpression; oxidation; photosynthetic bacteria; programs; protein expression; protein function; protein structure; receptor; research study; respiratory; sample fixation; small molecule libraries; stigmatellin; structural biology; success; tool; trans-Golgi Network; ubiquinol; uptake; virtual

Over the past few years, we have determined the structures of the cytochrome bc1 complex from 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 suggested 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 can be used to verify this hypothesis; and this is an ongoing process. (i) We were able to crystallize Rsbc1 only in the presence of Pf inhibitors such as stigmatellin, JG144 and famoxadone. (ii) The significance of the extraordinarily high sequence conservation of the cd1 helix and the PEWY motif in the ef loop of the cyt b subunit can now be appreciated, as both motifs contribute to the ISP binding surface. (iii) Based on our hypothesis, the b hemes should be reduced before the c1 heme, which were experimentally observed both from reports in the literature and in our measurement under pre-steady state conditions. (iv) In contrast to the previous hypothesis that the conformational fixation of ISP in the presence of stigmatellin was due to a H-bond between the bound inhibitor and ISP, we found that the elimination of this H-bond still promotes immobility of ISP in the cases of famoxadone or JG144 binding. These findings strongly support the idea that the switch of the ISP conformation is an intrinsic property of the bc1 complex. If this H-bond were indeed important for the conformational fixation of ISP, the QH2/Q would have served as an inhibitor rather than a substrate, as the same H-bond exists between bound Q/QH2 and ISP. (v) Mutagenesis of residues contributing to the ISP binding surface on cyt b invariably changes ET kinetics between ISP and c1 but does not affect QH2 binding to the QP site. (vi) For Rsbc1, the I292M and I292A mutants that have significantly slowed down ET between ISP and c1 can be crystallized in the absence of Pf inhibitors. Our current studies focus on investigating the conformational transition of ISP in the absence of respiratory inhibitors. We have discovered that ISP conformation switch induced by redox potentials changes. 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 and is 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. 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 3.5 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.

在过去的几年里，我们确定了来自光合细菌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。转为解释对苯二酚氧化（QP）位点的分叉电子转移（ET），提供了实验证据来支持这一假设，并确定了底物泛醇（QH2）首次登陆 QP 网站。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的结构研究表明，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。文献报道和新实验的数据可以用来验证这一假设；这是一个持续的过程。 (i) 只有在 Pf 抑制剂（例如豆豆菌素、JG144 和 famoxadone）存在的情况下，我们才能使 Rsbc1 结晶。 (ii) 现在可以认识到 cd1 螺旋和 cyt b 亚基 ef 环中 PEWY 基序的极高序列保守性的重要性，因为这两个基序都有助于 ISP 结合表面。 (iii) 根据我们的假设，b 血红素应该在 c1 血红素之前减少，这是从文献报告和我们在稳态前条件下的测量中实验观察到的。 (iv) 与之前的假设相反，即在豆黄素存在下 ISP 的构象固定是由于结合的抑制剂和 ISP 之间的氢键造成的，我们发现这种氢键的消除仍然促进了 ISP 在famoxadone 或 JG144 结合的情况。这些发现有力地支持了这样的观点，即 ISP 构象的转换是 bc1 复合物的固有特性。如果该氢键对于 ISP 的构象固定确实很重要，则 QH2/Q 将充当抑制剂而不是底物，因为结合的 Q/QH2 和 ISP 之间存在相同的氢键。 (v) 有助于 cyt b 上 ISP 结合表面的残基的诱变总是会改变 ISP 和 c1 之间的 ET 动力学，但不影响 QH2 与 QP 位点的结合。 (vi) 对于 Rsbc1，显着减慢 ISP 和 c1 之间 ET 的 I292M 和 I292A 突变体可以在不存在 Pf 抑制剂的情况下结晶。我们目前的研究重点是研究在没有呼吸抑制剂的情况下 ISP 的构象转变。我们发现氧化还原电位变化引起ISP构象转换。 自 20 世纪 70 年代获得 FDA 批准以来，顺铂化疗已成为广泛癌症治疗的基石，也是当今癌症医学中最常用的化疗药物之一。与许多其他化疗药物一样，顺铂在临床应用中面临着日益严重的癌症耐药问题。已经提出了多种顺铂耐药性产生的机制，包括细胞摄取和药物流出的变化、药物解毒的增加、细胞凋亡的抑制和DNA修复的增加。尽管该领域进行了广泛的研究，但顺铂耐药的分子机制仍然难以捉摸。假设的蛋白质 TMEM205（以前称为 MBC3205）被分泌蛋白发现计划推测为分泌性整合膜蛋白。该蛋白质由 189 个氨基酸残基和四个预测的跨膜螺旋 (TMH) 组成。人们对这种蛋白质及其细胞功能知之甚少，直到最近我们报道了它在癌细胞系顺铂耐药性中的作用。使用荧光标记的顺铂，结果表明TMEM205的过度表达可减少癌细胞系中顺铂的积累；这种减少与细胞的顺铂耐药性相关。 TMEM205 是一种定位于细胞表面的膜蛋白，在人顺铂耐药宫颈癌和肝癌细胞内部靠近跨高尔基体网络中高表达。该蛋白在某些分泌组织中存在高表达，例如肝脏、胰腺和肾上腺，这与 TMEM205 在顺铂耐药性中的假设作用一致。为了获得 TMEM205 的结构解决方案，我们过表达了这种整合膜蛋白，确定了其寡聚状态，并将其结晶。 TMEM205 晶体将 X 射线衍射至 2 A 分辨率。多药耐药性（MDR）是癌症治疗中长期存在的临床挑战。 MDR 与细胞表面 P-糖蛋白 (P-gp) 等外排 ABC 转运蛋白的过度表达有关。在癌症治疗期间停止 P-gp 的努力尚未成功。 我的实验室长期致力于阐明P-gp的结构，试图从结构角度揭示P-gp的功能机制。最近，我们在酵母表达系统中成功表达了人和小鼠的P-gp。更重要的是，我们能够结晶小鼠 P-gp 并将小鼠 P-gp 晶体衍射至 3.5 A 分辨率。这一成功为我们提供了研究人类和小鼠 P-gp 溶液行为差异的机会，并有可能提供更好的小鼠 P-gp 结构。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。