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)。它包括大小从小分子到整个蛋白质结构域的溶质的出口商和进口商。 真核生物 ABC 转运蛋白主要挤出疏水性分子 (5)，而大多数细菌 ABC 转运蛋白则输入必需的营养物质。 ABC 转运蛋白的功能单元由两个具有核苷酸 (ATP) 结合和水解结构域（NBD 或 ATP 盒）的分子马达组成，每个分子马达与编码底物特异性决定因素的跨膜结构域 (TMD) 偶联，并提供结合室和跨膜通道。 ABC 转运蛋白四个结构域的分子组织是从许多 ABC 输入蛋白 (6-9) 以及细菌多药物外排系统 Savl866(^0) 和 MsbA (11) 的晶体结构中收集的。 ABC 出口商的一个子类与多重耐药性 (MDR) 有关。人 P-糖蛋白 (Pgp) 和来自乳酸乳球菌的 LmrA 能够挤出多种药物分子；前者为肿瘤细胞提供了逃避化疗 6 种药物毒性的策略{12-14)。 该桥接项目旨在回答该领域的一个重大悬而未决的问题，即表征将 ATP 能量输入转换为药物运输的构象运动的性质和幅度。细菌 ABC 输出蛋白 Sav1866 和 MsbA (10, 11) 的最新晶体结构，以及脂质体中 MsbA 的广泛自旋标记分析 (15-18) 定义了诱导的构象变化的蓝图 通过 ATP 结合。然而，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”二态模型与 Pgp 的生化分析不兼容，Pgp 生化分析识别出运输循环中至少六种中间体（19）。缺失的环节是了解 ABC 转运蛋白在转运中间体之间循环时的构象动力学。这个问题的本质要求 用于研究 ABC 转运蛋白在其天然环境中的结构的方法，具有足够的空间分辨率和动态灵敏度来连接结构和功能。 Pgp 为功能动力学光谱分析提供了一个理想的系统。除了其直接的医学意义外，还积累了大量描述其与底物相互作用的信息，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 的自旋标记分析（20）。最近确定的无核苷酸 {apo) Pgp {21) 的晶体结构为实验设计和动力学计算研究提供了极好的起点，并为解释光谱数据提供了背景。 Pgp 被捕获为面向内的构象 其中两个对称相关的半部，每个半部由六个螺旋组成，被包装成V形几何形状，导致对细胞质和双层的内部小叶开放的空腔（图1B）。晶体结构还精确定位了水/膜界面附近的假定药物进入端口，允许药物进入空腔。 这种结构在机械上被解释为准备结合药物的预运输状态。 这种结构引发了许多重要问题。很可能 apo Pgp 需要对一个大的构象集合进行采样以适应光谱 运输的基材。这些构象之一通过晶格中的接触选择性地稳定。因此，晶体结构是否捕获了最多的人口 需要测试膜中的构象异构体。转运的底物会刺激 ATP 酶活性，并且它们的结合预计会通过诱导信号向 NBD 发出信号 构象变化（22）。然而，Pgp 的底物结合晶体结构实际上没有观察到任何变化，这进一步增强了对构象选择性的担忧{23)。 鉴于这些问题。目标 1 重点确定结晶的 apo 结构是否反映了膜中的平均构象并定义了构象 各种类型的 Pgp 底物引起的变化。我们还将确定空腔的可达性并分析假定的入口门户中的环境，如下 底物结合。 如果 apo 状态确实向细胞质开放，则预计 ATP 结合和水解将导致实质性的结构重排。这些的蓝图可以从 MsbA 和 Sav1866 的核苷酸结合结构中收集（图 1A）。这两个 NBD 构成了 典型的 ATP 三明治； TMD 经历交替进入，其中空腔靠近细胞质和内部双层小叶，并向细胞外侧开放。这种重新配置的基础是细胞质侧的大距离变化和跨膜螺旋的广泛重新包装。为了创建细胞外开口，扭转运动重新包装 TM 螺旋，从而改变 MsbA 两半之间交换的螺旋的身份。我们基于包含 MsbA {11) 结构的 AMPPNP 生成了人类 Pgp 的完全能量最小化同源模型，其处于向外的构象中（图 1A）。假设apo小鼠Pgp的新结构(ABCB1a)(23)代表处于向内构象的Pgp(图1B)，则预测在多药物转运过程中这两个关键状态之间会发生大幅构象变化(图1C) 。目标 2 和 3 建议测试在存在各类 Pgp 底物的情况下以 MsbA 为中心的 ATP 诱导构象变化模型。 下面描述的研究将有助于对 Pgp 介导的多药物外流现象进行分子描述。将定义和测试其他分析方法无法获得的结构中间体。关键功能中间体的结构和动力学以及它们之间构象变化的性质最终可能为Pgp功能特定调节剂的合理设计提供模板。