Phospholipid Metabolism in Cell Membrane

描述（由申请人提供）：磷脂代谢是细胞的基础。它不仅产生基本的生物膜，而且在几乎所有组织的细胞信号传导过程中起着重要作用。此外，许多蛋白质，无论是球状的还是膜结合的，都需要特定的磷脂来完成它们的功能。细胞维持着一个复杂而受调控的代谢网络，以合成多种磷脂并及时降解以满足细胞的需要。磷脂代谢的许多步骤发生在细胞膜上，并由膜内酶催化。由于结构信息的缺乏，人们对其分子机制知之甚少。特别是，这些酶如何从脂质膜双分子层中选择底物并在疏水膜环境中进行催化是一般磷脂代谢机制尚未解决的核心问题。为了理解这个重要的问题，我们研究了脂质磷酸磷酸酶（LPPs）作为模型。LPPs是膜内磷酸酶蛋白家族的成员，在磷脂合成和体内平衡中发挥重要作用。LPPs还催化一些重要的磷脂激素信使的去磷酸化，调节许多磷脂介导的信号传导过程。基于我们最近从大肠杆菌中获得的LPP同源物PgpB蛋白的载脂蛋白形态晶体结构，我们提出了一种关于PgpB膜内去磷酸化机制的新假设，其中a)磷脂底物从膜双分子层进入一个保守的膜内通道到达催化位点，b) TM3的大构象变化是底物结合和催化所必需的。为了验证这个重要的假设，在这个项目中，我们将集中在两个关键方面，使用生化，生物物理和x射线结构方法的组合。1)为了证明底物结合构象和底物诱导的蛋白质构象变化，我们将确定与代谢稳定的磷脂底物类似物或磷酸盐产物类似物钒酸盐结合的PgpB复合物结构，以获得催化循环的结构细节。2)为了从功能上表征膜内底物通道，我们设计了几种诱变和交联策略来阐明底物如何通过膜内通道到达催化位点。我们还将使用类似的方法探索产物释放途径，以了解催化后去磷酸化产物如何传递到膜双分子层。3)为了进一步证明蛋白质的构象变化，我们将应用EPR和荧光停流方法来捕捉蛋白质在洗涤剂溶液或不同脂质纳米圆盘中响应底物类似物结合的原子细节运动。这些结构和功能的研究不仅证实了我们的假设，揭示了膜内磷脂去磷酸化的催化机制，而且为全面了解细胞膜内磷脂代谢奠定了结构基础。

DESCRIPTION (provided by applicant): Phospholipid metabolism is fundamental in cells. It not only generates basic biological membranes, but also plays important roles in cellular signaling processes in nearly all tissues. In addition, many proteins, both globular and membrane bound, require specific phospholipids to fulfill their functions. Cells maintain a complicated and regulated metabolic network to synthesize a great diversity of phospholipids and degrade them in a time fashion to meet cellular requirements. Many steps of phospholipid metabolism take place on the cell membrane and are catalyzed by membrane-embedded enzymes. Their molecular mechanisms are poorly understood largely due to the paucity of structural information. In particular, how these enzymes select their substrates from the lipid membrane bilayer and carry out catalysis in a hydrophobic membrane environment is a central question still unanswered for general phospholipid metabolic mechanisms. To understand this important question, we study lipid phosphate phosphatases (LPPs) as model. LPPs, members of an intramembrane phosphatase protein family, play important roles in phospholipid synthesis and homeostasis. LPPs also catalyze dephosphorylation of several important phospholipid hormonal messengers regulating numerous phospholipids-mediated signaling processes. Based on our recent apo form crystal structure of the PgpB protein, an LPP homolog from E.coli, we proposed a novel hypothesis for the intramembrane dephosphorylation mechanism of PgpB, in which a) phospholipid substrates access an conserved intramembrane tunnel from the membrane bilayer to reach the catalytic site and b) a large conformational change of TM3 is essential for substrate binding and catalysis. To test this important hypothesis, in this project w will focus on two key aspects using a combination of biochemical, biophysical and X-ray structural approaches. 1) To demonstrate the substrate binding conformation and substrate-induced protein conformational changes, we will determine PgpB complex structures bound with a metabolism-stabilized phospholipid substrate analog or vanadate, a phosphate product analog, to gain structural details of a catalytic cycle. 2) To functionally characterize the intramembrane substrate access tunnel, we have designed several mutagenesis and crosslinking strategies to elucidate how the substrate passes through the intramembrane tunnel to reach the catalytic site. We will also explore the product release pathway using similar approaches to understand how the dephosphorylated product is delivered to the membrane bilayer after catalysis. 3) To further demonstrate the protein conformational changes, we will apply EPR and fluorescence stopped-flow approaches to catch the protein motions in response to the substrate analog binding in atomic detail in detergent solutions or in different lipid-defind nanodiscs. These structural and functional studies will not only confirm our hypothesis and reveal the catalytic mechanism of intramembrane phospholipid dephosphorylation, but also establish a structural basis to understand phospholipid metabolism in the cell membrane in general.