Structural basis of integral membrane enzyme function

摘要 脂类的合成和修饰主要是由至少部分嵌入到 双层结构本身。这些酶反应不仅对所有细胞膜的生物合成至关重要， 也用于脂质介导的信号传递和将作为脂质结合物的可溶性分子输出到细胞外 广泛的基本细胞功能的隔间，包括蛋白质和脂肪糖基化，以及 外膜化学性质的改变，作为细胞对变化的适应 环境。然而，尽管我们对膜蛋白功能的了解取得了进展，我们的 膜酶如何与其脂质底物在分子水平上相互作用的知识一直是 稀有，也受到脂类成分本身产生的疏水性的阻碍。 我的实验室的主要重点是使用结构生物学在分子水平上研究相互作用 在膜酶和它们的脂类底物之间。我们的结构将产生可测试的功能 关于疏水和亲水底物如何发生催化作用的假说， 关于涉及带电基团和水环境的化学反应如何适应过程 疏水配体底物专一性的分子决定因素， 以及膜本身在这些过程中所起的作用。我们期待在以下方面达成共同原则 膜、膜酶和脂质底物之间的相互作用将从我们的研究中显现出来。 我们最初的注意力集中在CDP-乙醇催化的甘油磷脂的生物合成上。 磷酸转移酶家族，以及糖的酶偶联和解偶联 由聚异戊二烯糖基转移酶GtrB和氨基阿拉伯糖转移酶Arnt组成的聚异戊二烯载体， 分别进行了分析。我们将继续沿着这些方向，通过获得这些酶的结构与 它们的脂类配体在模拟脂类双层环境中，通过x-射线结晶学在脂类立方体中 在脂类填充的纳米盘中，可以通过单粒子冷冻电子显微镜(Cryo-EM)来检测相变(LCP)。我们还将 在新的方向上扩展，通过合成和修改 革兰氏阴性杆菌的脂多糖成分(O-抗原连接酶、乙醇胺 转移酶EPT A和ARNT)，通过将活化的糖偶联到聚异戊二烯载体(GtrB和Dolicol- 磷酸甘露糖合成酶(DPMS)，并通过糖-聚异戊二烯的解偶联生成 成熟内毒素(Waal)，修饰脂质A(ARNT)，或糖基化蛋白(Pomt1/2)。 为了取得成功，我们将结合我们在膜蛋白生产、生物化学和结构方面的专业知识 生物学，到我们的合作者，他们是各自领域的领导者，从化学合成 糖脂结合物，内毒素的生化分析，膜蛋白的功能分析 从重组系统或动物模型到工具的生成，以允许对小蛋白进行冷冻-EM分析。

ABSTRACT Lipids are synthesized and modified primarily by integral membrane enzymes embedded, at least in part, in the bilayer itself. These enzymatic reactions are essential not only for the biosynthesis of all cellular membranes, but also for lipid-mediated signaling and for the export of soluble molecules as lipid conjugates to outer cellular compartments for a wide array of basic cellular functions, which include protein and lipid glycosylation, and modifications of the chemical properties of outer membranes as an adaptation of the cell to a changing environment. However, despite the advances in our understanding of how membrane proteins function, our knowledge of how membrane enzymes interact with their lipidic substrates at a molecular level has been scarce, also hindered by the hydrophobicity engendered by the lipid constituents themselves. The main focus of my lab is to use structural biology to investigate at a molecular level the interactions between membrane enzymes and their lipidic substrates. Our structures will produce testable functional hypotheses on how hydrophobic and hydrophilic substrates are brought into apposition for catalysis to occur, on how chemical reactions involving charged groups and an aqueous environment can adapt to process lipophilic molecules, on what are the molecular determinants of substrate specificity for hydrophobic ligands, and on the role that the membrane itself plays in these processes. We expect common principles on the interactions between, membrane, membrane enzymes, and lipidic substrates to emerge from our studies. We have focused our initial attention on glycerophospholipid biosynthesis as catalyzed by the CDP-alcohol phosphotransferase family of enzymes, and on the enzymatic coupling and uncoupling of sugars to polyisoprenyl carriers by the polyisoprenyl glycosyltransferase GtrB and the aminoarabinose transferase ArnT, respectively. We will continue in these directions by obtaining structures of these enzymes in complex with their lipidic ligands in mimics of the lipid bilayer environment, either by x-ray crystallography in lipidic cubic phase (LCP), or by single-particle cryo-electron microscopy (cryo-EM) in lipid-filled nanodiscs. We will also expand in new directions, related one with the other by the synthesis and modification of the lipopolysaccharide (LPS) component of Gram-negative bacteria (O-antigen ligase WaaL, ethanolamine transferase Ept A, and ArnT), by the coupling of activated sugars to polyisoprenyl carriers (GtrB and dolichol- phosphate mannose synthase, DPMS), and by the uncoupling of sugar-polyisoprenyl conjugates to generate mature LPS (WaaL), to modify lipid A (ArnT), or to glycosylate proteins (Pomt1/2). To succeed, we will combine our expertise in membrane protein production, biochemistry, and structural biology, to that of our collaborators that are leaders in their respective fields, ranging from chemical synthesis of sugar-lipid conjugates, to biochemical analysis of LPS, to functional analyses of membrane proteins in reconstituted systems or animal models, to the generation of tools to allow cryo-EM analysis of small proteins.