Structural Analysis of Biological Membrane Proteins

多药耐药（MDR）是癌症治疗和微生物感染治疗中长期存在的临床挑战。耐多药的一个主要原因是外排ABC转运体如p糖蛋白（P-gp）在细胞表面的过度表达。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P-gp的原子分辨率结构解析，试图从结构角度揭示P-gp的作用机制。最近，我们通过x射线晶体学成功地确定了稳定的小鼠P-gp的各种形式的结构，为我们分析P-gp功能的结构基础提供了能力。更重要的是，这一成功为我们提供了一个机会来研究人类和小鼠P-gp溶液行为的差异，这可能会导致人类P-gp的结构解决方案。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。测定了牛线粒体（Mtbc1）和光合细菌sphaeroides （Rsbc1）细胞色素bc1复合体的不同形态结构，提出了表面亲和调节铁硫蛋白（ISP）构象转换的机制假说，以解释喹啉氧化（QP）位点的分叉电子转移（ET），并提供了实验证据支持这一假说。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。

Multidrug resistance (MDR) is a long-standing clinic challenge in cancer therapies and in treatment of microbial infections. A major cause of MDR is the 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 elucidation of the structure at atomic resolution 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 determined structures of a stabilized mouse P-gp in various forms by X-ray crystallography, which afford us the ability to analyze 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 potentially may lead to the structure solution of human 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. 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.