STRUCTURAL AND FUNCTIONAL ANALYSIS OF GLUCOSE TRANSPORTERS

描述（由申请人提供）：本提案的总体目标是以原子分辨率确定葡萄糖转运蛋白（GLUT）功能的结构基础。这将在分子水平上揭示其运输机制以及参与糖尿病，癌症和其他疾病的病理学。它也将为开发新药和医学检测方法提供一个合理的平台。GLUT携带碳水化合物（葡萄糖，果糖，半乳糖等），活细胞的主要能量来源，穿过膜。到目前为止，已经鉴定和表征了14种人GLUT。尽管它们具有相对高的序列相似性，但它们在功能和组织表达方面表现出显著的变异性。确定任何GLUT的三维（3D）结构将是研究这一基本运输系统的一个重大突破，将开辟新的研究途径。结合定点突变和功能研究的结构信息将使我们能够查明GLUT成员的功能变异性的分子基础，从而可以合理地追求为特定GLUT设计的配体。为了了解葡萄糖转运蛋白的功能多样性并增加获得结构解决方案的机会，目标包括：人葡萄糖转运蛋白（hGLUT）和与hGLUT氨基酸序列具有大于25%同一性和45%同源性的几种细菌对应物。产生了毫克量的用于七种细菌和三种人GLUT的纯化的功能性蛋白。到目前为止，结晶努力已经成功地与四个细菌GLUT，其中两个产生衍射晶体;晶体的一个细菌GLUT衍射高达3.1埃分辨率。 我们的假设是：1）细菌GLUT的三维结构将代表人类GLUT的三维结构，因为活性位点残基从原核到真核物种都是保守的; 2）作为MFS成员，GLUT具有两个对称的6-螺旋束和一个亲水性内腔，在转运机制期间经历构象变化。我们的两个具体目标是：1a）确定产生衍射良好晶体的细菌GLUT的3D结构。1b）产生至少一种纯化的人GLUT的衍射晶体。2a）研究纯化的GLUT的转运。2b）开发GLUT的高通量结合分析以寻找新的抑制剂。我们提出的研究结果将影响我们对GLUT的基本理解。此外，它们将以两种方式使寻找用于治疗涉及GLUT的疾病的新药成为可能：1）GLUT的结构确定将指导合理的药物设计; 2）通过小分子的高通量筛选来鉴定新型配体将为新药提供先导化合物。

DESCRIPTION (provided by applicant): The overarching aim in this proposal is to determine at atomic resolution the structural basis of glucose transporter (GLUT) function. This will reveal, at a molecular level, its transport mechanism and involvement in pathologies of diabetes, cancer and other diseases. It will also provide a rational platform for developing both novel drugs and medical detection methods. GLUT carries carbohydrates (glucose, fructose, galactose and others), major energy sources for living cells, across membranes. So far fourteen human GLUTs have been identified and characterized. They exhibit significant variability in function and tissue expression despite their relatively high sequence similarity. Determining the three-dimensional (3D) structure of any GLUT will be a remarkable breakthrough in the study of this fundamental transport system that wil open new research avenues. Structural information in conjunction with site-directed mutagenesis and functional studies will allow us to pinpoint the molecular base of the functional variability of GLUT members, so that designed ligands for a particular GLUT can be rationally pursued. To understand the functional diversity of glucose transporters and increase the chances of obtaining a structural solution, the targets include: human glucose transporters (hGLUTs), and several bacterial counterparts with greater than 25% identity and 45% homology to hGLUTs amino-acid sequences. Milligram quantities of purified, functional protein for seven bacterial and three human GLUTs were produced. Thus far, crystallization efforts have been successful with four bacterial GLUTs among which two produced diffracting crystals; crystals of one bacterial GLUT diffracted up to 3.1 Angstroms resolution. Our hypotheses are that 1) the 3D structure of bacterial GLUTs will be representative for those of human GLUTs, as the active site residues are conserved from prokaryotic to eukaryotic species, and 2) as a MFS member, GLUT has two symmetric 6-helicies bundles and a hydrophilic internal cavity which undergo conformational changes during the transport mechanism. Our two specific aims are to: 1a) Determine the 3D structures of those bacterial GLUTs that yield well-diffracting crystals. 1b) Produce diffracting crystals of at least one purified human GLUT. 2a) Investigate the transport of purified GLUTs. 2b) Develop high-throughput binding assay for GLUTs in order to find novel inhibitors. The results of our proposed studies will impact our fundamental understanding of GLUTs. Furthermore, they will empower the search for novel drugs for the treatment of diseases involving GLUTs in two ways: 1) structure determination of GLUTs will guide rational drug design; 2) identification of novel ligands through high- throughput screening of small molecules will provide lead compounds for new drugs.