Structural Basis of Biological Membrane Protein Functions and Drug Resistance

细胞耐药性是由几种机制引起的。从结构的角度来看，我的团队将我们的研究集中在两个最常见的：靶位点突变和细胞内药物浓度的降低。靶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bc 1复合物（Btbc 1，也称为细胞呼吸链的复合物III）和光合细菌R的结构和功能。各种形式的sphaeroides（Rsbc 1）。复合物III是针对病原微生物的抗生素的经验证的靶标。基于我们的结构和功能的研究，我们提出了一个假设，以解决中央机制问题的Q-循环机制的复杂III的功能。这一假说，被称为表面亲和力调制的铁硫蛋白（ISP）构象开关，解决了在醌氧化（QP）的细胞色素bc 1复合物的网站的分叉电子转移（ET）的机制。通过对不同抑制剂作用下的Rsbc 1结构的测定，进一步证实了铁硫蛋白在不同抑制剂作用下的构象转换。十多年来的广泛研究可以说解决了关于细胞色素bc 1复合物的结构-功能关系的大多数问题，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，包括TCA循环的组分如苹果酸脱氢酶（MDH）对细胞色素bc 1的调节，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。我们也一直在研究复合物III的生物发生，通过阐明bcs 1的结构，bcs 1是一种线粒体膜蛋白，在协助ISP亚基插入复合物III的核心组装中至关重要。细胞内药物浓度的降低代表了耐药性的另一个重要机制。多药耐药（Multidrug resistance，MDR）是肿瘤化疗和微生物感染治疗中长期存在的临床难题，是指肿瘤或病原微生物对多种无关治疗药物同时产生耐药或交叉耐药。MDR的机制之一是细胞表面外排ABC转运蛋白如人P-糖蛋白（hP-gp）的过度表达。逆转hP-gp的功能以克服肿瘤治疗中的MDR的前景推动了P-gp特异性抑制剂的开发。然而，这些努力迄今为止都是不成功的，尽管广泛的研究旨在阐明这些P-gp抑制剂的功能的潜在机制。一个问题显然与缺乏P-gp的详细结构知识有关，这些知识与P-gp的催化途径中的各个沿着步骤有关，解决方案是获得与这些抑制剂复合的hP-gp的结构，从而可以揭示详细的相互作用。作为第一步，我们必须获得天然形式和各种构象的hP-gp的结构。我的实验室长期以来一直致力于在原子分辨率下解析hP-gp和mP-gp（小鼠P-gp）的结构，最近又致力于ZfP-gp（斑马鱼P-gp），我们试图从结构的角度揭示P-gp功能的机制，特别是它们识别结构多样性化合物的能力。我们希望解决的一些问题是（1）理解P-gp底物多特异性的结构基础，（2）ATP水解与底物易位的偶联，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，该灵活性由连接P-gp的两个半部分的75个残基的接头促进。我们缩短了接头，以促进mP-gp的结构测定，随后用于许多其他mP-gp结构的成功结构测定。这些结构导致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，接头缩短的突变体P-gp具有基础的ATP酶活性，但失去了药物刺激的ATP酶活性。(2)接头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在全长P-gp中观察到的相同的面向内的构象，这表明接头缩短的突变体的功能丧失是由于蛋白质的柔性丧失。(3)在没有底物的情况下，P-gp仅在NBD 1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)一系列的野生型，接头突变体，和甲基化的P-gp的结构分析表明，个别跨膜结构域螺旋的P-gp进行显着的运动，这是非常重要的，密切相关的开放和关闭运动的两个叶的P-gp。因此，P-gp的两个半部分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一个机会，以研究人类和小鼠P-gp之间的溶液行为的差异，这，正如我们所希望的那样，可能会导致hP-gp的结构解决方案。

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.