Dynamics and mechanism of sodium-dependent carboxylate transporters

钠依赖性羧酸转运蛋白的动力学和机制

• 批准号: 10577283
• 负责人: Ruben L Gonzalez
• 金额: $ 68.39万
• 依托单位: NEW YORK UNIVERSITY SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-01-22 至 2026-11-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10577283
• 关键词: Anions; Bacteria; Binding; Binding Sites; Biochemical; Biological Assay; Caloric Restriction; Cell membrane; Charge; Citrates; Compensation; Complement; Computer Models; Coupled; Coupling; Cryoelectron Microscopy; Cytoplasm; Data; Development; Diabetes Mellitus; Drug Industry; Elevator; Energy-Generating Resources; Event; Family; Family member; Fatty Acids; Fatty acid glycerol esters; Fluorescence Resonance Energy Transfer; Free Energy; Glycolysis; Homologous Gene; Human; Individual; Industry; Insulin Resistance; Ions; Kinetics; Knockout Mice; LDL Cholesterol Lipoproteins; Lipids; Liposomes; Malignant Neoplasms; Measurement; Measures; Membrane; Membrane Transport Proteins; Metabolic; Metabolic Diseases; Methods; Molecular; Molecular Conformation; Mutation; Nature; Obesity; Pathogenesis; Pathway interactions; Play; Positioning Attribute; Productivity; Proteins; Reaction; Role; Sampling; Sequence Homology; Series; Side; Site; Sodium; Specificity; Structure; Succinates; Techniques; Testing; Time; Work; carboxylate; carboxylation; citrate carrier; design; dicarboxylate-binding protein; driving force; drug development; experimental study; extracellular; fatty acid biosynthesis; fly; improved; inhibitor; member; molecular dynamics; nanodisk; oxidation; protein transport; proteoliposomes; prototype; sensor; single-molecule FRET; small molecule inhibitor; sodium ion; stoichiometry; structural determinants; symporter; therapeutic target

PROJECT SUMMARY The plasma membrane-bound sodium-dependent citrate transporter (NaCT) plays a major role in fatty acid biosynthesis and thus represents a major therapeutic target for various metabolic diseases. Its bacterial homolog, VcINDY, is a prototype of the entire divalent anion sodium symporter (DASS) family, of which NaCT is also a member. Despite the enormous progress that has been made, major gaps remain in our current understanding of the molecular mechanisms of NaCT, VcINDY, and other secondary membrane transporters. In particular, we do not know how a transporter works in real-time. At best, mechanistic descriptions of the transport cycles of these transporters consist of a series of structural snapshots of some of the individual states, along with limited kinetic parameters connecting the various states obtained from ensemble measurements. In this project, we aim to characterize the structure, dynamics, function, and inhibition of these two sodium-driven carboxylate transporters. (1) We will first characterize the structural basis of sodium-substrate coupling in VcINDY and NaCT. We will identify the ion stoichiometry and the positions and structures of the unknown sodium sites in the transporters. Following measurements of sodium-dependent substrate binding in solution, we will determine the structures of the transporters in the presence and absence of sodium using cryo-EM. Any hypothesis suggested by such structures will be examined by biochemical experiments. (2) Using smFRET, we will characterize the real-time dynamics with which VcINDY and NaCT transition between the outward- and inward-facing states during transport. Specifically, we will identify the number of conformational states that are sampled during transport and determine the rates of transitions between those states. Using this framework, we will investigate how the sodium ions and substrates that generate the driving force for transport modulate these parameters. These experimental measurements will be integrated into computational models generated by MD simulations. (3) We will aim to understand the entire reaction cycle of the transporter, including the energy landscape. We will develop FRET-based succinate and citrate sensors to measure the transport activities of individual transporters in single liposomes. These measurements will be used to construct the entire free-energy landscape of the transporter using MD simulations. (4) Finally, we will elucidate the mechanisms of inhibition of NaCT by several classes of allosteric inhibitors from the pharmaceutical industry to improve their potency and specificity in order to help design better strategies in the treatment of metabolic disorders.

项目摘要 质膜钠依赖性柠檬酸转运蛋白（NaCT）在脂肪酸代谢中起着重要作用， 生物合成，因此代表了各种代谢疾病的主要治疗靶点。其细菌 同源物VcINDY是整个二价阴离子钠同向转运体（DASS）家族的原型，其中NaCT是 也是一名成员。尽管取得了巨大进展，但我们目前的工作仍存在重大差距。 了解NaCT，VcINDY和其他二级膜转运蛋白的分子机制。在 特别是，我们不知道传输器如何实时工作。充其量，对运输的机械描述 这些转运体的循环由一系列单个状态的结构快照组成，沿着 用有限的动力学参数连接从系综测量获得的各种状态。在这 项目，我们的目标是表征这两个钠驱动的结构，动力学，功能和抑制 羧酸转运蛋白。(1)我们将首先描述钠-基质耦合的结构基础， VcINDY和NaCT。我们将确定未知钠的离子化学计量以及位置和结构 运输机上的位置在测量溶液中的钠依赖性底物结合之后，我们将 使用cryo-EM确定存在和不存在钠时转运蛋白的结构。任何 这种结构所提出的假设将通过生物化学实验来检验。(2)使用smFRET，我们 将表征VcINDY和NaCT在外向和外向之间转换的实时动态特性。 在运输过程中的内向状态。具体来说，我们将确定构象状态的数量， 在传输期间采样并确定这些状态之间的转换速率。利用这个框架，我们 将研究钠离子和底物如何产生运输的驱动力，调节这些 参数这些实验测量将被整合到由MD生成的计算模型中。 模拟(3)我们的目标是了解运输机的整个反应周期，包括能量 景观我们将开发基于FRET的琥珀酸盐和柠檬酸盐传感器来测量 单个脂质体中的单个转运蛋白。这些测量将用于构建整个自由能 景观的运输机使用MD模拟。(4)最后，我们将阐明抑制的机制， NaCT通过来自制药工业的几类变构抑制剂来提高其效力， 特异性，以帮助设计更好的治疗代谢紊乱的策略。