Structure of Cation-Coupled Active Sugar Transporters

由电化学离子梯度驱动的主动运输（即，次级主动运输）是在所有生命形式中发现的基本生物过程，其在细胞功能的许多方面（例如营养吸收、信号传导和对环境中有害成分的抵抗）中起着重要作用。本项目的目的是获得大肠杆菌蜜二糖通透酶（MelB）的X射线结构。最近，在这一总的领域取得了突破。两个二级转运蛋白，LacY和GlpT的X-射线结构进行了测定。然而，没有任何Na+偶联的二级转运蛋白的X射线晶体结构。MelB由469个氨基酸残基组成，并且是糖苷-戊糖苷-己糖醛酸苷：阳离子共转运体家族的生物化学充分研究的成员。MelB通过利用来自Na+、Li+或H+的能量向下向内运动的自由能来驱动半乳糖苷糖的化学计量向上运输来催化吡喃半乳糖苷的积累，并且耦合离子取决于所运输的糖的性质。通常，α-半乳糖苷（蜜二糖、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-吡喃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成为高度独特的转运蛋白。像LacY一样，MelB似乎含有12个跨膜结构域，N-和C-末端面向细胞质。已经获得了二维晶体，低分辨率的投影图显示了一个由两个结构域组成的分子，这两个结构域排列在一个中央裂缝中，类似于LacY的整体结构;然而，无论是糖的位置还是阳离子结合位点都没有得到解决，使得Na+驱动的运输机制没有得到解决。最近，在共结晶过程中磷脂的浓度的操纵已经导致了可重复的晶体LacY，其可重复作用到更高的分辨率，以及一些重要的突变体缺陷的H+易位。通过使用所描述的方法，已经获得了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组分，其结合MelB和LacY并与甘油激酶共结晶。这反映在蛋白质数据库（PDB）中有大约40，000个可溶性蛋白质结构，但只有大约45个独立的膜蛋白质结构。了解阳离子耦合传输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，这些活动预计将导致利用H+或Na+的独特转运体的X射线结构。预期的结果将显着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，以获得和改善膜蛋白晶体的质量是一种新的方法，磷脂效应的进一步表征可以提供一个基本的指导方针，膜蛋白结晶的一般方法，这仍然是结构生物学的主要障碍。更广泛的影响：LacY是一种专性的H+/糖同向转运体，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。这些研究报告被广泛挑选，以多种语文纳入世界各地本科生和研究生教学的各种教科书、参考书和教学材料。该项目将涉及向高中和大学的受众进行宣传，以便向年轻人传播科学知识，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，供公众通过向公共数据库提交原始数据访问。邀请讲座，在研讨会上的口头报告，在世界各地举行的多学科会议将作为多种渠道，及时向社会传达这一新知识。

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.