Structure of Cation-Coupled Active Sugar Transporters

阳离子偶联活性糖转运蛋白的结构

• 批准号: 0450970
• 负责人: Ronald Kaback
• 金额: $ 132.73万
• 依托单位: University of California-Los Angeles
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-05-15 至 2011-04-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0450970&HistoricalAwards=false
• 关键词: Structure; Cation; Coupled; Active; Sugar

Active transport driven by electrochemical ion gradients (i.e., secondary active transport) is a fundamental biological process found in all life forms that plays an essential role in many aspects of cell function, such as nutrient uptake, signal transduction and resistance to noxious components in the environment. The aim of this project is to obtain an x-ray structure of the melibiose permease of Escherichia coli (MelB). Recently, a breakthrough was achieved in this general area. X-ray structures of two secondary transporters, LacY and the GlpT, were determined. However, there is no x-ray crystal structure available for any Na+-coupled secondary transport protein. MelB consists of 469 amino acid residues and is a biochemically well-studied member of the glycoside-pentoside-hexuronide:cation symport family. MelB catalyzes the accumulation of galactopyranosides by utilizing the free energy from the energetically downhill inward movement of Na+, Li+ or H+ to drive stoichiometric uphill transport of galactosidic sugars, and the coupling ion is dependent on the nature of the sugar transported. Generally, alpha-galactosides (melibiose, raffinose and p-nitrophenyl-alpha-galactoside) are symported with either H+ or Na+, while beta-galactosides (lactose, methyl-1-D-galactopyranoside or p-nitrophenyl-beta-D-galactoside) are symported with Na+ but not with H+, which makes MelB a highly unique transporter. Like LacY, MelB appears to contain 12-transmembrane domains with the N- and C-termini facing the cytoplasm. 2D crystals have been obtained, and a projection map at low resolution displays a molecule consisting of two domains lining a central cleft, similar to the overall structure of LacY; however, neither the location of the sugar nor the cation binding sites are resolved, making the mechanism of Na+-driven transport unresolved. Lately, manipulation of the concentration of phospholipids during co-crystallization has led to reproducible crystals of LacY that diffract to higher resolution, as well as a number of important mutants defective in H+ translocation. By using the approaches described, initial crystals of MelB have been obtained. The structure anticipated will provide important insights. Cocrystallization will also be attempted with IIA-Glc, a soluble regulatory component of the phosphoenolpyruvate:sugar phosphotransferase system that binds to MelB and LacY and co-crystallizes with glycerol kinase.Membrane proteins, particularly those that catalyze ion-coupled transport, are notoriously difficult to crystallize presumably because of their hydrophobic nature and conformational flexibility. This is reflected by the fact that there are some 40,000 structures of soluble proteins in the Protein Data Bank (PDB), but only about 45 independent membrane protein structures. Understanding of the mechanism of the cation coupled transport is a major challenge in the field of bioenergetics. This project extends a recent breakthrough, and these activities are expected to lead to an x-ray structure of a unique transporter that utilizes either H+ or Na+. The expected results will significantly improve overall knowledge of active transport and bioenergetics. In addition, manipulation of phospholipid concentrations to obtain and improve the quality of membrane protein crystals is a novel approach and further characterization of the phospholipid effect may provide a basic guideline for a general method of membrane protein crystallization, which remains a major barrier in structural biology. Broader Impacts: Structure/function studies on LacY, an obligate H+/sugar symporter, have served as a model for studies on active transport and bioenergetics. These studies have been widely selected for inclusion in various textbooks, reference books and teaching materials in many languages for both undergraduate and graduate teaching worldwide. The project will involve outreach to high school and college audiences in order to convey scientific knowledge to young people and stimulate their interest in basic science. The progress expected will directly provide databases for public access by submission of original data to the PDB. Invited lectures, oral presentations at symposia, multi-disciplinary conferences held around the world will serve as multiple channels to convey this novel knowledge to society in a timely manner.

由电化学离子梯度驱动的主动转运（即二次主动转运）是所有生命形式中发现的一个基本生物过程，在细胞功能的许多方面起着至关重要的作用，如营养摄取、信号转导和对环境中有害成分的抗性。本项目的目的是获得大肠杆菌（MelB）的糖二糖渗透酶的x射线结构。最近，在这方面取得了突破。测定了LacY和GlpT两种次级转运体的x射线结构。然而，没有任何Na+偶联的二次转运蛋白的x射线晶体结构。MelB由469个氨基酸残基组成，是糖苷-戊糖-己糖醛酸阳离子共配家族的一个生物化学研究的成员。MelB通过利用Na+、Li+或H+的下坡运动产生的自由能来驱动半乳糖糖的化学计量上坡运输，从而催化半乳糖苷的积累，并且偶联离子取决于所运输的糖的性质。一般来说，α -半乳糖苷（糖苷、棉子糖和对硝基苯基- α -半乳糖苷）与H+或Na+共转运，而β -半乳糖苷（乳糖、甲基-1- d -半乳糖苷或对硝基苯基- β - d -半乳糖苷）与Na+共转运，但不与H+共转运，这使得MelB成为一种非常独特的转运体。与LacY一样，MelB似乎包含12个跨膜结构域，其N和c端面向细胞质。已经获得了二维晶体，在低分辨率的投影图上显示了一个分子，由两个区域组成，排列在中心裂缝中，类似于LacY的整体结构；然而，糖和阳离子结合位点的位置都没有确定，使得Na+驱动转运的机制没有确定。最近，在共结晶过程中对磷脂浓度的操纵导致了可复制的LacY晶体衍射到更高的分辨率，以及一些重要的H+易位缺陷突变体。采用上述方法，获得了MelB的初始晶体。预期的结构将提供重要的见解。还将尝试与IIA-Glc共结晶，IIA-Glc是磷酸烯醇丙酮酸：糖磷酸转移酶系统的可溶性调节成分，与MelB和LacY结合，并与甘油激酶共结晶。众所周知，膜蛋白，特别是那些催化离子偶联运输的膜蛋白，很难结晶，这可能是因为它们的疏水性和构象灵活性。这反映在蛋白质数据库（Protein Data Bank， PDB）中约有40,000种可溶性蛋白结构，但只有约45种独立的膜蛋白结构。了解阳离子耦合输运的机制是生物能量学领域的一个重大挑战。该项目扩展了最近的一项突破，这些活动有望导致利用H+或Na+的独特转运体的x射线结构。预期的结果将显著提高主动运输和生物能量学的整体知识。此外，操纵磷脂浓度来获得和提高膜蛋白晶体的质量是一种新方法，进一步表征磷脂效应可能为膜蛋白结晶的一般方法提供基本指导，这仍然是结构生物学的主要障碍。更广泛的影响：LacY是一种专性H+/糖同调转运体，其结构/功能研究已成为主动转运和生物能量学研究的模型。这些研究已被广泛选择纳入各种教科书、参考书和多种语言的教学材料中，供世界各地的本科和研究生教学使用。该项目将涉及向高中和大学观众进行宣传，以便向年轻人传达科学知识并激发他们对基础科学的兴趣。预期的进展将直接提供数据库供公众查阅，方法是将原始数据提交给数据数据库。邀请讲座、专题讨论会的口头报告、在世界各地举行的多学科会议将成为及时向社会传达这一新知识的多种渠道。