Phospholipid Metabolism in Cell Membrane

描述(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过程中都扮演着重要的角色。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和受调控的代谢网络，以合成各种磷脂，并以一种时间的方式将其降解，以满足细胞的需求。磷脂代谢的许多步骤都发生在细胞膜上，并由膜包埋的酶催化。其分子机制在很大程度上是由于缺乏结构信息而知之甚少。特别是，这些酶如何从脂膜双层中选择底物并在疏水性膜环境中进行催化，是一般磷脂代谢机制仍未回答的中心问题。为了理解这个重要的问题，我们研究了脂磷酸盐磷酸酶(LPP)作为模型。LPPS是膜内磷酸酶家族的成员，在磷脂合成和动态平衡中发挥重要作用。LPPS还催化几个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过程。根据我们最近对PgpB蛋白的apo形式的晶体结构，我们对PgpB的膜内去磷酸化机制提出了一个新的假说，其中a)磷脂底物从膜双层进入保守的膜内隧道到达催化位置，b)TM3的大构象变化是底物结合和催化所必需的。为了验证这一重要的假设，在这个项目中，w将结合使用生化、生物物理和X射线结构方法，重点关注两个关键方面。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产物类似物钒酸结合的PgpB复杂结构，以获得催化循环的结构细节。2)为了确定膜内底物通道的功能，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置。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是如何输送到膜双层的。3)为了进一步证明蛋白质构象的变化，我们将应用EPR和荧光停流方法来捕捉洗涤剂溶液或不同脱脂纳米盘中底物类似物原子细节结合时蛋白质的运动。这些结构和功能的研究不仅证实了我们的假说，揭示了膜内磷脂去磷酸化的催化机制，而且为从总体上理解细胞膜中的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.