Structure of Cation-Coupled Active Sugar Transporters

由电化学离子梯度驱动的主动转运(即二次主动转运)是一种存在于所有生命形式中的基本生物学过程，它在细胞功能的许多方面发挥着重要作用，如营养物质的吸收、信号转导和对环境中有害成分的抵抗等。本项目的目的是获得大肠杆菌(MELB)中二糖渗透酶的X射线结构。最近，这一总体领域取得了突破。测定了Lacy和GlpT两种次级转运蛋白的X射线结构。然而，对于任何钠离子偶联的次级运输蛋白，都没有可用的X射线晶体结构。MELB由469个氨基酸残基组成，是糖苷-戊苷-己糖醛酸阳离子转运蛋白家族中生物化学研究较多的成员。Melb通过利用Na+、Li+或H+的能量下坡向内运动的自由能来催化半乳糖苷的积累，从而驱动半乳糖苷的化学计量上行运输，而耦合离子依赖于所运输的糖的性质。一般来说，α-半乳糖苷(蜂蜜二糖、棉籽糖和对-硝基苯基-α-半乳糖苷)与H+或Na+共存，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+共存，但不与H+共存，这使得Melb成为一种非常独特的转运蛋白。与Lacy一样，Melb似乎含有12个跨膜结构域，N-末端和C-末端面向细胞质。已经获得了2D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，排列在一个中心裂隙上，类似于Lacy的整体结构；但是，糖的位置和阳离子结合位置都没有得到解决，使得Na+驱动的转运机制没有得到解决。最近，在共结晶过程中对磷脂浓度的控制已经导致了可重复的Lacy晶体，这些晶体衍射到更高的分辨率，以及一些在H+转运方面有缺陷的重要突变体。利用所述方法，获得了MALB的初始晶体。预期的结构将提供重要的见解。还将尝试用IIA-GLC进行共结晶，IIA-GLC是磷酸烯醇式丙酮酸的一种可溶性调节成分，它与Melb和Lacy结合并与甘油激酶共结晶。膜蛋白，特别是那些催化离子偶联运输的蛋白质，众所周知很难结晶，可能是因为它们的疏水性和构象灵活性。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，这一事实反映了这一点。了解阳离子耦合输运的机理是生物能量学领域的一大挑战。该项目延续了最近的一项突破，预计这些活动将导致一种独特的利用H+或Na+的转运体的X射线结构。预期的结果将显著提高对主动运输和生物能量学的整体知识。此外，控制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，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，膜蛋白结晶仍然是结构生物学中的主要障碍。更广泛的影响：对专性H+/糖转运体Lacy的结构/功能研究已成为主动运输和生物能量学研究的模型。这些研究已经被广泛地选为各种教科书、参考书和多种语言的教材，供全世界的本科生和研究生教学使用。该项目将与高中和大学的受众进行接触，以便向年轻人传播科学知识，并激发他们对基础科学的兴趣。预计的进展将通过向PDB提交原始数据直接提供数据库，供公众查阅。在世界各地举办的特邀讲座、研讨会上的口头陈述、多学科会议将成为向社会及时传达这一新奇知识的多个渠道。

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.