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 质膜结合的钠依赖性柠檬酸盐转运蛋白（NACT）在脂肪酸中起主要作用 生物合成，因此代表了各种代谢疾病的主要治疗靶点。它的细菌 同源物，vcindy是整个二价阴离子钠钠（dass）家族的原型，其中nact是 也是成员。尽管取得了巨大进展，但我们的当前仍存在重大差距 了解NACT，VCINDY和其他次级膜转运蛋白的分子机制。在 特别是，我们不知道运输商如何实时工作。充其量是运输的机械描述 这些转运蛋白的周期由某些单个状态的一系列结构快照组成， 有限的动力学参数连接从集合测量获得的各种状态。在这个 项目，我们旨在表征这两个钠驱动的结构，动力学，功能和抑制 羧酸盐转运蛋白。 （1）我们将首先表征在钠含量耦合的结构基础 vcindy and nact。我们将确定离子化学计量学以及未知钠的位置和结构 转运蛋白的站点。在测量溶液中钠依赖性底物结合的测量之后，我们将 在存在和不存在冷冻EM钠的情况下，确定转运蛋白的结构。任何 这些结构提出的假设将通过生化实验进行研究。 （2）使用smfret，我们 将表征vcindy和向外和裸体过渡的实时动力学 运输过程中向内的状态。具体来说，我们将确定构象状态的数量 在运输过程中采样并确定这些状态之间的过渡速率。使用此框架，我们 将研究产生运输驱动力的钠离子和底物如何调节这些驱动力 参数。这些实验测量将集成到由MD生成的计算模型中 模拟。 （3）我们将旨在了解转运蛋白的整个反应周期，包括能量 景观。我们将开发基于FRET的琥珀酸酯和柠檬酸盐传感器，以测量 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 使用MD模拟的转运蛋白的景观。 （4）最后，我们将阐明抑制的机制 从制药行业的几类变构抑制剂裸露以提高其效力和 特异性是为了帮助设计更好的策略来治疗代谢疾病。