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

几乎每个分子穿过细胞膜的过程都是由一类被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是至关重要的，当这些过程受到干扰或中断时，就会发生各种疾病，比如几种遗传疾病或肿瘤细胞上调多药耐药转运蛋白。获得人类转运蛋白的高分辨率结构对于确定其机制的分子结构基础是至关重要的。我们建议建立一个膜蛋白结构测定中心TransportPDB，旨在开发一种全面而有效的方法来追踪目前在人类中发现的48个家族的521个转运蛋白的高分辨率X射线晶体结构，以及PSI生物中心的其他靶点。为此目的并针对防扩散安全倡议的目标，我们有以下具体目标： A1.一条高效的流水线将建立在成熟技术的基础上，并利用我们的经验成功地结晶和解决完整的膜蛋白的X射线结构。这条管道将基于几个主要原则：(A)根据疾病相关性确定靶标优先顺序，并完成人类转运蛋白的蛋白质折叠空间覆盖；(B)专用真核表达系统(毕赤酵母和293S哺乳动物细胞)，这些系统已被证明能提供适合结晶的功能蛋白质；(C)克隆基于合成基因的结构，该合成基因针对这两个表达系统的表达进行了优化；以及(D)使用最先进的数据收集技术来适度衍射晶体。这个漏斗状的组织将能够筛选数百个人类转运蛋白目标及其密切的哺乳动物同源基因，朝着成功获得它们的X射线晶体结构的目标迈进。 A2.将开发高通量的方法和技术，用于靶标的功能和生物物理表征，以快速确定保持蛋白质稳定性和功能的条件，从而获得更高质量和更好的衍射性人类转运蛋白晶体。将实施新的晶体安装方法，以及微束/光栅化技术和提高数据收集的灵敏度(Pilatus探测器)，这可能对适度衍射膜蛋白晶体起决定性作用。 A3.建立结构和功能数据以及对科学界有用的其他材料的资源，包括人类转运蛋白的X射线晶体结构、密码子优化的克隆、洗涤剂的增溶条件以及每个靶标的相应稳定性和同源模型。

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.