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

E.大肠杆菌二酰基甘油激酶（DAGK）是一种同源三聚体（40 kDa，123个残基亚基）的膜蛋白，它将ATP的γ-磷酸转移到脂质二酰基甘油（DAG）上，并将其转化为磷脂酸（PA）。脂质底物和脂质产物在不同的信号通路中起着重要的作用。DAGK属于一类重要的脂质修饰酶，其催化在水相和膜相中发生的反应。DAGK在序列和结构方面不同于其他激酶。它不显示通常在催化磷酰基转移反应的其他酶中发现的序列基序，并且没有可用的结构同源性模型，因为该蛋白质家族在结构上非常多样。因此，本提案的具体目的是使用多核稳态以及时间分辨MAS-NMR光谱，辅以cwDNP和EPR光谱，直接在脂质双层内解决DAGK在分子水平上的功能机制。固态NMR为解决膜界面的酶促机制提供了巨大的潜力。该项目所建立的结构、动力学和动力学研究方法也将适用于其他脂质调节剂的研究，这是理解脂质信号分子基础的重要前提。

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.