TransportPDB: Center for the X-ray Structure Determination of Human Transporters

事实上，每一个分子穿过细胞膜都是由一类称为转运蛋白的蛋白质介导的。转运蛋白对所有细胞的生物学都至关重要，当这些过程受到干扰或破坏时，就会发生各种疾病，如几种遗传疾病或肿瘤细胞上调多药耐药转运蛋白。人类转运蛋白的高分辨率结构的可用性对于定义其机制的分子结构基础是必不可少的。我们建议建立一个中心的膜蛋白结构的测定，TransportPDB，与开发一个全面的和有效的方法，追求高分辨率的X射线晶体结构的521转运蛋白在48个家庭目前确定的人类和其他目标从PSI生物中心。为此目的，并为了实现《防扩散安全倡议》的目标，我们有以下具体目标： A1.将基于成熟的技术和我们成功结晶和解决完整膜蛋白的X射线结构的经验建立一个有效的管道。这一管道将基于几个关键原则：（a）基于疾病相关性和完成人转运蛋白的蛋白质折叠空间覆盖的靶标优先化，（B）真核表达系统的专门使用（巴斯德毕赤酵母和293 S哺乳动物细胞），其已被证明递送适合于结晶的功能性蛋白，（c）基于优化用于在两种表达系统中表达的合成基因的构建体的克隆，和（d）使用最先进的数据收集技术用于适度衍射晶体。漏斗状组织将能够筛选数百个人类转运蛋白靶点及其密切的哺乳动物直系同源物，从而实现成功获得其X射线晶体结构的目标。 A2.将开发高通量方法和技术，用于靶点的功能和生物物理表征，以快速确定维持蛋白质稳定性和功能的条件，从而实现更高质量和更好的衍射人类转运蛋白晶体。新的晶体安装方法，连同微束/光栅技术和提高灵敏度的数据收集（PILLENT检测器），将实施可能是决定性的适度衍射膜蛋白质晶体。 A3.建立对科学界有用的结构和功能数据以及其他材料的资源，包括人类转运蛋白的X射线晶体结构、密码子优化克隆、去污剂溶解条件以及每个靶标和同源模型的相应稳定性特性。

The passage of virtually every molecule across the cell membrane is mediated by a class of proteins called transporters. Transporters are vital to the biology of all cells and a variety of diseases occur when these processes are perturbed or disrupted, as in several genetic disorders or the up-regulation of multidrug resistance transporters by tumor cells. The availability of high resolution structures of human transporters is essential to define the molecular structural basis of their mechanisms. We propose to establish a center for membrane protein structure determination, TransportPDB, with the objective of developing a comprehensive and efficient approach for pursuing the high-resolution x-ray crystal structures of 521 transporters in 48 families presently identified in humans and other targets from PSI-biology centers. For this purpose and to address the objectives of the PSI, we have the following specific aims: A1. An efficient pipeline will be established based on proven technologies and using our experience successfully crystallizing and solving the x-ray structures of integral membrane proteins. This pipeline will be based on several key principles: (a) target prioritization based on disease relevance and completing the protein-fold space coverage of human transporters, (b) the exclusive use of eukaryotic expression systems (Pichia pastoris and 293S mammalian cells) that have proven to deliver functional protein suitable for crystallization, (c) the cloning of constructs based on synthetic genes optimized for expression in both expression systems, and (d) using state-of-the-art data collection techniques for modestly diffracting crystals. The funnel-like organization will enable screening hundreds of human transporter targets and their close mammalian orthologs, driving towards the goal of successfully obtaining their x-ray crystal structures. A2. High-throughput methods and technology will be developed for functional and biophysical characterization of targets to rapidly identify conditions that maintain protein stability and function leading towards higher quality and better diffracting human transporter crystals. New crystal mounting methods, together with micro-beam/rastering technology and increased sensitivity in data collection (PILATUS detector), will be implemented that could be decisive for modestly diffracting membrane protein crystals. A3. Establish a resource for structural and functional data and other materials useful to the scientific community, including x-ray crystal structures of human transporters, codon-optimized clones, detergent solubilization conditions and corresponding stability properties of each target and homology models.