Structual Characterization of the Na+/Glucose Cotransporter Family

描述（由申请人提供）：膜转运蛋白控制能量转导，修饰离子浓度，并主动将代谢物导入细胞。此外，国际上最广泛使用的两种药物——氟西汀和奥美拉唑——针对的是膜转运蛋白。一类重要的转运蛋白是Na+/葡萄糖共转运蛋白家族（SGLT），它包含240多个成员，存在于所有生命领域。SGLT有11种人同种异构体，在多种组织中表达。编码Na+/糖同向转运体SGLT1和SGLT2以及Na+/碘化物共转运体（NIS）的至少三个基因的突变已知会导致代谢紊乱。这些蛋白质在生理学中发挥着重要作用，并与许多疾病有关，最明显的是感染性腹泻、糖尿病和某些癌症。考虑到这些蛋白质的功能和药理相关性，从这个共转运蛋白家族中获得三维晶体结构，将极大地促进我们对这些共转运蛋白如何工作的理解，并使我们能够了解它们在健康、疾病和治疗中的作用。我们对来自副溶血性弧菌（vSGLT）的Na+/半乳糖共转运体有初步的晶体学数据。这些晶体在Na+和半乳糖的存在下生长，目前衍射到4.5埃，这些晶体的优化正在进行中。利用SGLT家族其他成员的信息以及糖和Na+结合位点的特征序列，我们已经确定了在运输中应该有影响的残基。我们将在这些残基上构建vSGLT突变体，并使用放射性摄取和荧光分析来分析它们。使用这些方法，我们将能够测量运输速率，对Na+和半乳糖的明显亲和力，以及糖特异性的变化。最终，这些生化和生物物理数据将结合vSGLT的原子分辨率结构进行分析。在获得vSGLT的结构并确定重要突变体后，我们将使用SGLT1、SGLT2和NIS同调子的骨干坐标进行分子线化，并利用现有的生化数据对模型进行完善。这些模型可能能够解释关于这些具有多种底物的多功能蛋白的一些关键问题，并可能促进药物设计。

DESCRIPTION (provided by applicant): Membrane transport proteins govern energy transduction, modify ion concentrations, and actively import metabolites into the cell. Moreover, two of the most widely prescribed drugs internationally-fluoxetine and omeprazole-target membrane transport proteins. One important class of transporters is the Na+/Glucose cotransporter family (SGLT), which contains over 240 members and is present in all kingdoms of life. There are eleven human isoforms of SGLT, expressed in a variety of tissues. Mutations in at least three of these genes, encoding the Na+/sugar symporters SGLT1 and SGLT2, as well as the Na+/iodide cotransporter (NIS), are known to result in metabolic disorders. These proteins perform essential roles in physiology and are implicated in a number of diseases, most notably infectious diarrhea, diabetes and some cancers. Given the functional and pharmacological relevance of these proteins, a 3D crystal structure from this family of cotransporters, would greatly advance our understanding of how these cotransporters work and enable us to understand their role in health, disease and therapy. We have preliminary crystallographic data on the Na+/galactose cotransporter from Vibrio parahaemolyticus (vSGLT). These crystals are grown in the presence of Na+ and galactose and are currently diffracting to 4.5Angstroms, and optimization of these crystals is in progress. Using information from other members of the SGLT family and signature sequences for sugar- and Na+-binding sites, we have identified residues that should be influential in transport. We will construct mutants of vSGLT at these residues and analyze them using radioactive uptake and fluorescence assays. Using these methods we will be able to measure transport rates, apparent affinities for Na+ and galactose, and alterations in sugar specificity. Ultimately, this biochemical and biophysical data will be analyzed in conjunction with the atomic resolution structure of vSGLT. After obtaining the structure of vSGLT and identifying important mutants, we will use the backbone coordinates for molecular threading of SGLT1, SGLT2 and NIS symporters and refine the models by using available biochemical data. These models may be able to explain some key questions about these multifunctional proteins with a diverse range of substrates and possibly facilitate drug design.