Structural Analysis of Biological Membrane Proteins

在过去的几年里，我们确定了光合细菌R. sphaeroides （Rsbc1）细胞色素bc1复合体的各种形式的结构，提出了表面亲和调节铁硫蛋白（ISP）构象转换的机制假设，以解释喹啉氧化（QP）位点的分节电子转移（ET），并提供了实验证据支持这一假设。首次在QP位点鉴定出底物泛醇（QH2）。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的结构解实现了我们通过结构、遗传和生化技术相结合，建立一个系统研究bc1复合体的模型体系的目标之一。我们对牛bc1的结构研究使我们提出，QP位点分叉ET的关键是控制ISP-ED的运动，这调节了2Fe2S簇和c1血红素之间的距离。当ISP-ED处于固定构象时，两个站点之间的距离太长而不允许ET；只有当ISP-ED处于移动构象时，ET才有可能实现。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。来自文献报告的数据以及我们实验室和其他人的新实验都支持这一假设。目前我们的重点是在实验中证明在没有抑制剂的情况下的控制机制，这更与生理条件有关。经过十多年的密集后3d结构研究，可以说已经解决了关于细胞色素bc1复合物的结构-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量学领域奠定了基础，包括TCA循环组分和分子氧对bc1的调节。分子氧使bc1的电子转移活性提高82%，这取决于配合物的完整性。在泛醇分岔氧化过程中，氧对细胞色素bc1复合物反应序列的影响发生在血红素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经通过共沉淀和它们增强bc1活性的能力得到证实。这些相互作用的结晶学研究正在进行中。自20世纪70年代获得FDA批准以来，顺铂化疗已成为广泛癌症治疗的基石，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，顺铂在临床应用中面临着日益严重的肿瘤耐药问题。对于顺铂耐药的发展，已经提出了许多机制，包括药物的细胞摄取和外排的变化、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。这个假设的蛋白TMEM205，以前被称为MBC3205，被secreted protein Discovery Initiative推测为一种分泌的整体膜蛋白。该蛋白由189个氨基酸残基和4个预测的跨膜螺旋（TMHs）组成。直到最近，当我们报道了它在癌细胞系顺铂耐药中的作用时，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，研究表明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耐药性有关。TMEM205是一种定位于细胞表面的膜蛋白，在人类顺铂耐药宫颈癌和肝癌细胞中高度表达，其内部靠近反式高尔基网络。在某些分泌组织中发现该蛋白的高表达水平，如肝脏、胰腺和肾上腺，这与TMEM205在顺铂耐药中的假设作用一致。为了获得TMEM205的结构溶液，我们过表达了该整体膜蛋白，测定了其寡聚态，并对其进行了结晶。TMEM205晶体衍射x射线至2a分辨率。目前，我们正在努力解决晶体相问题。多药耐药（MDR）是癌症治疗中一个长期存在的临床挑战。MDR与外排ABC转运蛋白如p糖蛋白（P-gp）在细胞表面的过度表达有关。在癌症治疗期间阻止P-gp的努力尚未成功。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的作用机制。最近，我们成功地在酵母表达系统中表达了人和小鼠P-gp。更重要的是，我们能够结晶小鼠P-gp和小鼠P-gp晶体衍射到3.5 A的分辨率。这一成功为我们研究人类和小鼠P-gp溶液行为的差异提供了机会，并有可能提供小鼠P-gp更好的结构。我的实验室还从事ABC转运体的分子建模研究，这已经成为了解原子结构未知的蛋白质结构和功能的重要工具。多年来，我们建立了ABCB1、ABCG2、Pdr5p等多种ABC转运蛋白的结构模型。这些模型是有用的指导，进一步表征这些蛋白质。

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 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 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. 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 3.5 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.