Bridging Project 2: Structural Dynamics of ABC Transporter

桥梁项目 2：ABC Transporter 的结构动力学

• 批准号: 7922843
• 负责人: Eduardo A Perozo
• 金额: $ 32.14万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-04-01 至 2015-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7922843
• 关键词: ABCB1 gene; ATP Hydrolysis; ATP phosphohydrolase; ATP-Binding Cassette Transporters; Adenylyl Imidodiphosphate; Antineoplastic Agents; Binding; Biochemical; Clinical; Coupled; Coupling; Cytoplasm; Cytotoxic agent; DNA Sequence Rearrangement; Data; Dimensions; Drug Efflux; Drug Transport; Effectiveness; Environment; Epilepsy; Excision; Excretory function; Experimental Designs; Family; Figs - dietary; Glean; Homology Modeling; Human; Human Pathology; Hydrolysis; Investigation; Kinetics; Lactococcus lactis; Lead; Light; Link; Liposomes; Malaria; Mediating; Medical; Membrane; Methods; Modeling; Molecular; Molecular Conformation; Molecular Models; Molecular Motors; Motion; Multi-Drug Resistance; Mus; Mycoses; Nature; Nucleotides; Nutrient; P-Glycoprotein; P-Glycoproteins; Pharmaceutical Preparations; Pharmacologic Substance; Physiological; Play; Process; Proteins; Resistance; Resistance development; Resolution; Role; Sampling; Shapes; Side; Signal Transduction; Spin Labels; Stroke; Structural Models; Structure; Substrate Specificity; System; Tertiary Protein Structure; Testing; Thermodynamics; Time; Toxic effect; Transmembrane Domain; Water; absorption; base; cancer therapy; chemotherapy; computer studies; conformer; cytotoxic; design; drug structure; efflux pump; extracellular; human disease; molecular dynamics; molecular modeling; multi drug transporter; multidrug transport; neoplastic cell; small molecule; solute

ATP-binding cassette (ABC) transporters constitute the largest family of transporters {1-4). It includes both exporters and importers of solutes ranging in size from small molecules to entire protein domains. Eukaryotic ABC transporters predominantly extrude hydrophobic molecules (5), while most bacterial ABC transporters import essential nutrients. The functional unit of an ABC transporter consists of two molecular motors with nucleotide (ATP) binding and hydrolysis domains (NBD or ATP cassette), each coupled to a transmembrane domain (TMD) that encodes the determinants of substrate specificity and provides the binding chamber and passageway across the membrane. The molecular organization of the four domains of ABC transporters was gleaned from crystal structures of a number of ABC importers {6-9) as well as the bacterial multidrug efflux systems Savl866(^0) and MsbA {11). A subclass of ABC exporters has been implicated in multidrug resistance (MDR). Human P-glycoprotein (Pgp) and LmrA from Lactococcus lactis are capable of extruding a large variety of drug molecules; the former providing a strategy for tumor cells to evade the toxicity of chemotherapeutic 6rugs{12-14). This Bridging Project aims to answer a major outstanding question in the field, namely to characterize the nature and amplitude of the conformational motions that transduce the ATP energy input to transport of drugs. Recent crystal structures of bacterial ABC exporters, Sav1866 and MsbA {10, 11), along with extensive spin labeling analysis of MsbA in liposomes {15-18) define a blueprint of the conformational changes induced by ATP binding. However, these investigations were carried out in the absence of drug or substrate. The resulting "minimalist" two-state model is not compatible with biochemical analysis of Pgp that identified at least six intermediates in the transport cycle {19). The missing link is an understanding of the conformational dynamics of ABC transporters as they cycle between transport intermediates. The nature of this problem calls for methods capable of investigating the structure of ABC transporters in their native environment with sufficient spatial resolution and dynamic sensitivity to link structure and function. Pgp provides an ideal system for spectroscopic analysis of functional dynamics. In addition to its direct medical significance, a wealth of information has been accumulated describing its interaction with substrates, including a detailed thermodynamic and kinetic analysis {19, 20). Furthermore, Dr. Al-Shawi has already initiated spin labeling analysis of Pgp {20). The recently determined crystal structure of nucleotide-free {apo) Pgp {21) provides an excellent starting point for experimental design and computational studies of the dynamics, and a context to interpret spectroscopic data. Pgp was captured in an inward-facing conformation where the two symmetry-related halves, each consisting of six helices, are packed in V-shaped geometry, resulting in a cavity open to the cytoplasm and the inner leaflet of the bilayer (Fig. IB). The crystal structure also pinpoints putative drug entry portals near the water/membrane interface that allow access to the cavity. This structure was interpreted mechanistically as a pre-transport state ready to bind drugs. This structure provokes a number of important questions. Very likely apo Pgp needs to sample a large conformational ensemble to accommodate the spectrum of transported substrates. One of these conformations is selectively stabilized by contacts in the crystal lattice. Thus, whether the crystal structure captures the most populated conformer in the membrane needs to be tested. Transported substrates stimulate the ATPase activity and their binding is expected to be signaled to the NBDs through induced conformational changes (22). However, virtually no changes were observed in the substrate-bound crystal structure of Pgp further reinforcing concerns of conformational selectivity {23). In light of these questions. Aim 1 focuses on determining whether the crystalized apo structure reflects the average conformation in the membrane and defines the conformational changes induced by various classes of Pgp substrates. We will also determine the accessibility of the cavity and analyze the environments in the putative entry portals following substrate binding. If indeed the apo state is open to the cytoplasm, ATP binding and hydrolysis are predicted to lead to substantial structural rearrangements. The blueprint of these can be gleaned from the nucleotide bound structures of MsbA and Sav1866 (Fig. 1A). The two NBDs form the canonical ATP sandwich; the TMDs undergo alternating access whereby the cavity closes to the cytoplasm and the inner bilayer leaflet and opens to the extracellular side. Underiying this reconfiguration are large distance changes on the cytoplasmic side and extensive repacking of transmembrane helices. To create the extracellular opening, a twisting motion repacks the TM helices changing the identity of the swapped helices between the two halves of MsbA. We generated a fully energy-minimized homology model of human Pgp in an outward-facing conformation based on the AMPPNP containing structure of MsbA {11) (Fig. 1A). Assuming that the new structure of apo mouse Pgp (ABCB1a)(23) represents Pgp in an inward-facing conformation (Fig. IB), large amplitude conformational changes are predicted between these two key states during multi-drug transport (Fig.lC). Aims 2 and 3 propose to test the MsbA-centric model of ATP-induced conformational change in the presence of the various classes of Pgp substrates. The investigations described below will facilitate a molecular description of the multi-drug efflux phenomenon mediated by Pgp. Structural intermediates inaccessible to other methods of analysis will be defined and tested. The structure and dynamics of key functional intermediates and the nature of conformational changes between them may eventually provide templates for the rational design of specific modulators of Pgp function.

ATP结合盒（ABC）转运蛋白构成最大的转运蛋白家族{1-4）。它包括从小分子到整个蛋白质结构域的溶质的出口商和进口商。 真核ABC转运蛋白主要是繁殖的疏水分子（5），而大多数细菌ABC转运蛋白会进口必需的营养。 ABC转运蛋白的功能单元由两个分子电动机组成，该电动机（ATP）结合和水解结构域（NBD或ATP盒）与跨膜域（TMD）耦合，该域（TMD）编码了底物特异性的确定因素，并在整个层次上编码了结合室和通道的确定性。 ABC转运蛋白四个结构域的分子组织是从许多ABC进口商{6-9）的晶体结构以及细菌多剂量外排系统SAVL866（^0）和MSBA {11）中收集的。 ABC出口商的子类与多药电阻（MDR）有关。乳酸乳球菌的人P-糖蛋白（PGP）和LMRA能够挤出大量的药物分子。前者为肿瘤细胞提供了逃避化学治疗6RUGS毒性的策略{12-14）。 这个桥接项目旨在回答该领域的一个主要问题，即表征将ATP能量输入到药物运输的构象运动的性质和振幅。细菌ABC出口商的最新晶体结构，SAV1866和MSBA {10，11），以及脂质体中MS​​BA的广泛自旋标记分析（15-18）定义了构象变化的蓝图。 通过ATP结合。但是，这些研究是在没有药物或底物的情况下进行的。由此产生的“极简主义”两态模型与PGP的生化分析不兼容，PGP在运输周期中至少识别了至少六个中间体（19）。缺失的链接是对ABC转运蛋白在运输中间体之间循环时的构象动力学的理解。这个问题的性质调用 对于能够在其天然环境中研究ABC转运蛋白结构的方法，并具有足够的空间分辨率和对链接结构和功能的动态敏感性。 PGP为功能动力学的光谱分析提供了理想的系统。除了其直接的医学意义外，还积累了大量信息，描述了其与底物的相互作用，包括详细的热力学和动力学分析{19，20）。此外，Al-Shawi博士已经启动了PGP {20）的自旋标记分析。最近确定的无核苷酸{Apo）PGP {21）的晶体结构为动力学的实验设计和计算研究提供了一个极好的起点，以及解释光谱数据的上下文。 PGP以向内的构象捕获 其中两个与对称性相关的半半螺旋组成的半几何形状堆积了，导致腔向细胞质和双层的内部小叶开放（图IB）。晶体结构还指出了允许进入腔的水/膜界面附近推定的药物进入门户。 这种结构被机理解释为准备结合药物的前运动状态。 这种结构引发了许多重要的问题。 Apo PGP很可能需要对大构象合奏进行采样以适应 运输的底物。这些构象之一通过晶体晶格中的接触选择性稳定。因此，晶体结构是否捕获了人口最多的 需要测试膜中的构象异构体。运输的底物刺激ATPase活性，预计其结合将通过诱导的 构象变化（22）。但是，在PGP的底物结合晶体结构中几乎没有观察到任何变化，进一步加强了构象选择性的关注点{23）。 鉴于这些问题。 AIM 1专注于确定晶体化的Apo结构是否反映了膜中的平均构象并定义了构象 各类PGP底物引起的变化。我们还将确定腔的可及性，并分析推定的入口门户中的环境 底物结合。 如果确实对Apo状态开放，则ATP结合和水解将导致实质性的结构重排。这些蓝图可以从MSBA和SAV1866的核苷酸结构结构中收集（图1A）。两个NBD形成 规范的ATP三明治； TMD交替进入，从而使腔接近细胞质和内部双层小叶，并打开细胞外侧。这种重新配置的不足是细胞质侧的距离发生较大的变化和跨膜螺旋的广泛重新包装。为了创建细胞外开口，扭曲运动重新包装TM螺旋改变了MSBA两半之间的交换螺旋的身份。我们基于包含MSBA {11）的AMPPNP的外向构象中的人类PGP的完全能量最小化的同源模型（图1A）。假设Apo小鼠PGP（ABCB1A）（23）的新结构代表向内构象中的PGP（图IB），则在多毒品传输过程中，这两个关键状态之间预测了较大的振幅构象变化（图.lc）。 AIM 2和3提出，在存在各种PGP底物的存在下，在存在ATP诱导的构象变化的以MSBA为中心的模型中。 下面描述的研究将促进对PGP介导的多药外排现象的分子描述。将定义和测试其他分析方法无法访问的结构中间体。关键功能中间体的结构和动力学以及它们之间的构象变化的性质最终可能为PGP功能特定调节器的合理设计提供模板。