Structural Analysis of Biological Membrane Proteins

我们测定了牛线粒体（Mtbc 1）和光合细菌R. sphaeroides（Rsbc1）的结构，提出了铁硫蛋白（ISP）构象转换的表面亲和性调节机制，解释了醌醇氧化（QP）位点的分叉电子转移（ET），提供了支持这一假说的实验证据，并首次鉴定了QP位点的底物泛醇（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的目标之一，在建立一个模型系统，系统地研究bc1复杂的结构，遗传和生物化学技术相结合。我们的牛bc1的结构研究使我们提出，在QP网站的分叉ET的关键是控制的ISP-ED的运动，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而不允许ET;只有当ISP-ED处于移动的构象时，ET才可能。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。来自文献报告和我们实验室和其他人的新实验的数据支持这一假设。目前，我们的重点是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。经过十多年的密集后3D结构研究可以说解决了大多数关于细胞色素bc1复合物的结构-功能关系的问题，为将这种重要复合物的知识整合到更广泛的生物能量学景观中奠定了基础，其中包括TCA循环和分子氧组分对bc1的调节。分子氧使bc1的电子转移活性提高了82%，这取决于配合物的完整性。氧对细胞色素bc1复合物的反应顺序的影响是在血红素bL还原的步骤中通过Q-循环机制氧化泛醇的分叉。TCA循环酶，苹果酸脱氢酶（MDH）和乌头酸酶（ACON）之间的特定相互作用，已被证明是由共沉淀和它们的能力，以提高bc1的活性。这些相互作用的晶体学研究正在进行中。自20世纪70年代获得FDA批准以来，顺铂化疗已成为广谱癌症治疗的基石，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，顺铂在其临床应用中面临着越来越多的癌症耐药问题。已经提出了许多顺铂耐药性的产生机制，包括细胞摄取和药物外排的变化，药物解毒作用的增加，细胞凋亡的抑制和DNA修复的增加。尽管在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MBC 3205，被推测为分泌蛋白质发现倡议的分泌整合膜蛋白。该蛋白由189个氨基酸残基组成，具有四个预测的跨膜螺旋（TMH）。直到最近我们报道了它在癌细胞系顺铂耐药中的作用，人们对这种蛋白及其细胞功能知之甚少。使用荧光标记的顺铂，显示TMEM205的过表达减少了顺铂在癌细胞系中的积累;这种减少与细胞的顺铂抗性相关。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中高度表达，位于trans-golgi网络附近。在某些分泌组织中发现该蛋白质的高表达水平，如肝、胰腺和肾上腺的分泌组织，这与TMEM 205在顺铂抗性中的假定作用一致。为了获得TMEM205的结构解决方案，我们过表达这种完整的膜蛋白，确定其寡聚体状态，并使其结晶。目前，我们正在努力解决晶体学相位问题。多药耐药（MDR）是肿瘤治疗中长期存在的临床难题。MDR与细胞表面外排ABC转运蛋白如P-糖蛋白（P-gp）的过度表达有关。在癌症治疗期间阻止P-gp的努力尚未成功。本实验室长期致力于P-gp的结构解析，试图从结构角度揭示P-gp的作用机制。最近，我们已经成功地表达人和小鼠P-gp在酵母表达系统。更重要的是，我们能够使小鼠P-gp结晶，并且小鼠P-gp的晶体衍射至3.0 A分辨率。这一成功为我们提供了一个机会，研究人类和小鼠P-gp的溶液行为的差异，并有可能提供一个更好的小鼠P-gp的结构。我的实验室还从事ABC转运蛋白的分子建模研究，这已经成为一个重要的工具，以获得结构和功能的见解蛋白质的原子结构是未知的。多年来，我们已经构建了一些ABC转运蛋白，如ABCB1，ABCG2，Pdr5p等的结构模型。这些模型是有用的指导，这些蛋白质的进一步表征。

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. 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 andis 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. Currently, we are working to solve the crystallographic phase problem. 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 3.0 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.