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。

• 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
• 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' 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

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， 由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）研究蛋白质与功能性脂质在双层环境，从蛋白脂质体， 生物膜这些测量将补充功能测定，荧光技术， 分子动力学（MD）模拟在相同的条件下。Kir通道参与长QT综合征， 低血糖症、巴特综合征、癫痫、药物滥用和周期性麻痹。Kir通道是配体 门控，但门控通道的结构和动力学的细节在很大程度上是未知的。Kir 2通道家族 由阴离子脂质磷脂酰肌醇4，5-二磷酸（PIP 2）门控，但由胆固醇失活， 与PIP 2竞争获取蛋白质。G蛋白激活的Kir通道（GIRK，Kir 3）通过与G蛋白的相互作用被门控。 PIP 2和Gbγ蛋白异源二聚体。在Kir 3家族中，胆固醇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，并量化所观察到的脂质的结构和动力学。 活动和非活动状态。我们将继续研究Kir通道KirBac1.1。我们分配了90%的 该蛋白质中的15 N和13 C化学位移（超过1600个独特的重原子），并使用这些分配来 识别变构，激活机制，与胆固醇二聚体结合的失活结构， 结构的封闭状态，并解决了结构的开放状态的通道。现在我们来测量 通道动态，并识别我们的数据中反映的通道的多个门控状态。我们将研究 在电压下的通道中的结构变化，并使用 冻结的动态核极化同时，我们还将研究Kir3.1-KirBac1.3通道 奇美拉初步数据确定了PIP 2结合残基和膜-水界面残基， 通道功能最终的目标将是哺乳动物Kir3.2（GIRK 2）通道及其完整的补体。 功能激活剂。在第二个项目中，我们将研究CC基序趋化因子受体CCR 3与CCL 11 脂质双层中的趋化因子。没有针对CCR 3的药物试验成功，这是不幸的，因为它涉及到 癌症转移、HIV进入和COVID 19细胞因子风暴。到目前为止，我们确定了CCL 11对接， 信号转导依赖于双层胆固醇剂量。初步的SSNMR研究发现胆固醇 构象选择受体的最佳配体结合构型。我们计划完全分配15 N 和胆固醇和阴离子脂质富集膜中CCR 3的13 C化学位移。这个结构 蛋白质与CCLL 11在不同的功能状态将得到解决，并测量区域动力学后， 为KirBac1.1建立了类似的工作流程。NMR也将用于解析CCL 11在溶液中的结构 并与CCR 3复合。我们将继续研究胆固醇低聚化，CCR 3二聚化， 这些事件之间。在整个过程中，我们将研究脂质寡聚化，动力学和蛋白质亲和力。