Integrated approaches to symport mechanisms of membrane transporters

膜转运蛋白转运机制的综合方法

• 批准号: 9895210
• 负责人: Lan Guan
• 金额: $ 4.42万
• 依托单位: TEXAS TECH UNIVERSITY HEALTH SCIS CENTER
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-07-01 至 2022-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9895210
• 关键词: 3-Dimensional; Active Biological Transport; Affinity; Automobile Driving; Binding; Biochemistry; Blood; Brain; Calorimetry; Cations; Coupled; Coupling; Crystallization; Disease; Docosahexaenoic Acids; Drug Targeting; Electron Spin Resonance Spectroscopy; Eye; Family; Free Energy; Galactosides; Genetic; Health; Homologous Gene; Human; Ion Cotransport; Knowledge; Ligand Binding; Ligands; Lysophosphatidylcholines; Measurement; Measures; Membrane; Membrane Transport Proteins; Molecular; Nerve Degeneration; Neurodegenerative Disorders; Pathway interactions; Play; Prevention; Protein Conformation; Retina; Role; Site; Solvents; Spin Labels; Structure; Testing; Titrations; United States National Institutes of Health; X-Ray Crystallography; base; improved; interdisciplinary approach; melibiose permease; nanobodies; neurodevelopment; novel; novel therapeutics; prevent; prototype; solute; success; symporter; targeted treatment; uptake

NIH 1R01GM122759-01A1 Integrated approaches to symport mechanisms of membrane transporters PROJECT SUMMARY/ABSTRACT Our long-term objective is to understand the molecular mechanisms of cation/solute symport catalyzed by membrane carriers. These transporters play critical roles in maintaining normal cellular activities, are important in human health and disease, and can serve as drug targets and therapeutic delivery pathways. In this proposal, we plan to study the bacterial Na+-coupled melibiose permease (MelB), which utilizes energy stored in the electrochemical gradient of Na+, Li+, or H+ to drive the translocation of galactoside against its concentration gradient, and is a prototype for exploring molecular mechanisms of symporters in the MFS family that can use more than one cationic species for coupling. The MelB homologue expressed in blood-brain and blood-retina barriers catalyzes Na+-coupled uptake of docosahexaenoic acids (DHA)-carrying lysophosphatidylcholine (LPC), thus supplying essential DHA to brain and eyes for neural development and prevents neurodegeneration. For secondary-active transport in general, the coupling between the driving cation and cargo solute is obligatory, but the mechanisms underlying the energetic coupling remain largely unknown. We will elucidate the Na+-coupled symport mechanisms by a combined approach, including genetics, biochemistry, calorimetry, site-directed spin labeling (SDSL) with continuous-wave electron paramagnetic resonance spectroscopy (CW-EPRs), and 3-D X-ray crystallography. We have created high- affinity MelB-camelid single-domain nanobodies (Nbs) for crystallization of MelB, and have implemented isothermal titration calorimetry (ITC) measurements to determine the free-energy changes and heat capacity changes for the binding of MelB’s ligands (melibiose, Na+, Li+), alone or together, as well as the CW-EPRs to measure ligand-induced solvent accessibility changes and proximity changes. Based on our strong preliminary results, three independent but complementary aims are proposed to test our central hypothesis: the core of the symport mechanism is cooperative binding of co-substrates that induces the formation of an occluded intermediate state. Our integrated multi-disciplinary approach will provide important missing information into the cation/solute symport mechanisms and improve our fundamental knowledge of the ligand binding energetics and protein conformational changes in general, as well as directly impact on other studies of Na+- coupled transporters including the LPC transporter in brain and retina.

国家卫生研究院1 r01gm122759-01a1