Resolving the functional mechanism of the lipid regulator diacylglycerolkinase by solid-state NMR

通过固态核磁共振解析脂质调节剂二酰甘油激酶的功能机制

基本信息

批准号：
237774529
负责人：
Professor Dr. Clemens Glaubitz
金额：
--
依托单位：
Institut für Biophysikalische Chemie
依托单位国家：
德国
项目类别：
Research Grants
财政年份：
2013
资助国家：
德国
起止时间：
2012-12-31 至 2017-12-31
项目状态：
已结题

来源：
https://gepris.dfg.de/gepris/projekt/237774529?language=en
关键词：
Resolving functional mechanism lipid regulator

项目摘要

E. coli diacylglycerolkinase (DAGK) is a homotrimeric (40 kDa, 123 residue subunits), integral membrane protein, which transfers the gamma-phosphate of ATP to the lipid diacylglycerol (DAG) converting it into phosphatidic acid (PA). Lipid substrate as well as lipid product play important roles in different signaling pathways. DAGK belongs to an important class of lipid modifying enzymes, which catalyze reactions taking place in both the aqueous and the membrane phase. DAGK is distinct from other kinases with respect to sequence and structure. It does not show sequence motifs typically found in other enzymes catalyzing phosphoryl transfer reactions and there is no structural homology model available as this protein family is very diverse in structure. The specific aim of this proposal is therefore to resolve the functional mechanism of DAGK at molecular level directly within the lipid bilayer using multinuclear steady-state as well as time-resolved MAS-NMR spectroscopy complemented by cwDNP and EPR spectroscopy. Solid-state NMR offers great potential to resolve enzymatic mechanisms at the membrane interface. The methodological toolkit for probing structure, dynamics and kinetics resulting from this project will be also applicable to other lipid regulators in the future, which is an important precondition to understand the molecular basis of lipid signaling.

大肠杆菌二酰基甘油酶（DAGK）是一种同二聚体（40 kDa，123个残基亚基），积分膜蛋白，将ATP的γ-磷酸γ转移到脂质二酰基甘油（DAG）中，将其转化为磷酸酸（PA）。脂质底物和脂质产物在不同的信号通路中起重要作用。 DAGK属于一类重要的脂质修饰酶，它们催化在水和膜相中发生的反应。在序列和结构方面，DAGK与其他激酶不同。它没有显示出在催化磷酸化转移反应的其他酶中通常发现的序列基序，并且由于该蛋白质家族的结构非常多样化，因此没有可用的结构同源模型。因此，该提案的具体目的是通过使用多核稳态以及时间分辨的MAS-NMR光谱来解决脂质双层在分子水平上的功能机制，并通过CWDNP和EPR光谱进行了补充。固态NMR具有解决膜界面上酶促机制的巨大潜力。该项目引起的探测结构，动力学和动力学的方法学工具包也将来适用于其他脂质调节剂，这是了解脂质信号传导的分子基础的重要前提。