The Functional Interplay of Lipid Membrane Components: Activation, Inhibition, and Raft Formation.

脂质膜成分的功能相互作用：激活、抑制和筏形成。

基本信息

批准号：
10623780
负责人：
Benjamin James Wylie
金额：
$ 49.84万
依托单位：
TEXAS TECH UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2017
资助国家：
美国
起止时间：
2017-08-10 至 2028-02-29
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10623780
关键词：
Affinity Alzheimer&apos s Disease Bartter Disease Binding Biochemical Biological Biological Assay CC chemokine receptor 3 CCL11 gene COVID-19 cytokine storm Chemicals Chimera organism Cholesterol Complement Complex Data Development Dimerization Disease Docking Dose Drug Addiction Environment Epilepsy Event Family Fluorescence Freezing Functional disorder G-Protein-Coupled Receptors GIRK2 subunit, G protein-coupled inwardly-rectifying potassium channel GTP-Binding Proteins Goals HIV Human Hypoglycemia Ligand Binding Lipid Bilayers Lipids Long QT Syndrome Measurement Measures Membrane Membrane Lipids Membrane Proteins Molecular Molecular Conformation Neoplasm Metastasis Nuclear Parkinson Disease Phosphatidylinositol 4,5-Diphosphate Phosphatidylinositols Potassium Channel Proteins Role Severities Signal Transduction Structure Substance abuse problem System Techniques Water autoinflammatory chemokine chemokine receptor dimer insight interfacial ligand gated channel molecular dynamics periodic paralysis protein structure proteoliposomes receptor solid state nuclear magnetic resonance targeted therapy trials voltage

项目摘要

Inward-rectifier K+ (Kir) channels and G protein-coupled receptors (GPCRs) are membrane proteins that are regulated by cholesterol and anionic lipids found in their native membranes. We will use solid-state NMR (SSNMR) to study proteins with functional lipids in bilayer environments ranging from proteoliposomes to biological membranes. These measurements will compliment functional assays, fluorescence techniques, and molecular dynamics (MD) simulations under identical conditions. Kir channels are involved in long-QT syndrome, hypoglycemia, Bartter’s syndrome, epilepsy, substance abuse, and periodic paralysis. Kir Channels are ligand gated, but details of the structure and dynamics of gated channels are largely unknown. The Kir2 channel family is gated by the anionic lipid phosphatidylinositol 4,5-bisphosphate (PIP2) but inactivated by cholesterol which competes with PIP2 to access the protein. G protein-activated Kir channels (GIRK, Kir3) are gated by the coaction of PIP2 and Gbγ protein heterodimers. In the Kir3 family, cholesterol increases rather than suppresses activity. Here we will explore the differing roles of functional lipids and quantify the structure and dynamics of the observed active and inactivated states. We will continue our studies of the Kir channel, KirBac1.1. We assigned 90% of the 15N and 13C chemical shifts in this protein (over 1600 unique heavy atoms) and used these assignments to identify allostery, the activation mechanism, the inactivated structure bound to a cholesterol dimer, refined the structure of the closed state, and solved the structure of the open state of the channel. Now we will measure the channel dynamics and identify the multiple gated states of the channel reflected in our data. We will study structural changes in the channel under voltage and identify discrete channel states and lipid contacts using freeze-trapped Dynamic Nuclear Polarization. In tandem, we will also study the Kir3.1-KirBac1.3 channel chimera. Preliminary data identifies PIP2 binding residues and membrane-water interfacial residues key for channel function. The eventual goal will be the mammalian Kir3.2 (GIRK2) channel and its full complement of functional activators. In a second project we will study the CC motif chemokine receptor CCR3 with the CCL11 chemokine in lipid bilayers. No drug trial targeting CCR3 has succeeded, which is unfortunate as it is involved in cancer metastasis, HIV entry, and the COVID19 cytokine storm. To date, we identified both CCL11 docking, and signal transduction are dose dependent upon bilayer cholesterol. Preliminary SSNMR studies found cholesterol conformationally selects for optimal ligand binding configurations of the receptor. We plan to fully assign the 15N and 13C chemical shifts of CCR3 in cholesterol and anionic lipid enriched membranes. The structures of this protein with CCLL11 in different functional states will be solved, and regional dynamics measured following a similar workflow established for KirBac1.1. NMR will also be used to solve the structures of CCL11 in solution and in complex with CCR3. We will pursue cholesterol oligomerization, CCR3 dimerization, and the relationship between these events. Throughout we will examine lipid oligomerization, dynamics, and protein affinity.

内向整流 K+ (Kir) 通道和 G 蛋白偶联受体 (GPCR) 是膜蛋白，受其天然膜中的胆固醇和阴离子脂质调节。我们将使用固态核磁共振。（SSNMR）研究双层环境中具有功能性脂质的蛋白质，从蛋白脂质体到这些测量将补充功能测定、荧光技术和相同条件下的分子动力学 (MD) 模拟 Kir 通道涉及长 QT 综合征，低血糖、巴特综合症、癫痫、药物滥用和周期性麻痹都是配体。门控通道，但门控通道的结构和动力学细节很大程度上未知。由阴离子脂质磷脂酰肌醇 4,5-二磷酸 (PIP2) 门控，但被胆固醇灭活，与 PIP2 竞争访问蛋白质 G 蛋白激活的 Kir 通道（GIRK、Kir3）通过相互作用进行门控。在 Kir3 家族中，PIP2 和 Gbγ 蛋白异二聚体的胆固醇会增加而不是抑制活性。在这里，我们将探讨功能性脂质的不同作用，并量化观察到的结构和动力学我们将继续研究 KirBac1.1 的 90%。该蛋白质中的 15N 和 13C 化学位移（超过 1600 个独特的重原子）并使用这些分配来确定变构、激活机制、与胆固醇二聚体结合的失活结构，精炼关闭状态的结构，并解决了通道打开状态的结构。现在我们将测量通道。我们将研究通道动态并识别数据中反映的通道的多个门控状态。电压下通道的结构变化，并使用以下方法识别离散通道状态和脂质接触同时，我们还将研究 Kir3.1-KirBac1.3 通道。初步数据确定了 PIP2 结合残基和膜-水界面残基的关键。最终目标将是哺乳动物 Kir3.2 (GIRK2) 通道及其完整补充。在第二个项目中，我们将研究 CC 基序趋化因子受体 CCR3 和 CCL11。脂质双层中的趋化因子尚未成功，这是不幸的，因为它涉及 CCR3。癌症转移、HIV 进入和 COVID19 细胞因子风暴迄今为止，我们确定了 CCL11 对接和信号转导依赖于双层胆固醇的剂量。初步 SSNMR 研究发现胆固醇。我们计划根据构象选择受体的最佳配体结合构型。胆固醇和阴离子脂质富集膜中 CCR3 的 13C 化学位移。将解决具有不同功能状态的 CCLL11 的蛋白质，并根据为 KirBac1.1 建立的类似工作流程也将用于解析溶液中 CCL11 的结构。以及与CCR3的复合物，我们将研究胆固醇寡聚化、CCR3二聚化以及它们之间的关系。在这些事件之间，我们将检查脂质寡聚、动力学和蛋白质亲和力。