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

TransportPDB：人类转运蛋白 X 射线结构测定中心

• 批准号: 8715818
• 负责人: GEOFFREY A CHANG
• 金额: $ 230.33万
• 依托单位: CALIFORNIA INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-30 至 2015-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8715818
• 关键词: Address; Automobile Driving; Biological Assay; Biology; Biomedical Research; Cell membrane; Cells; Cloning; Codon Nucleotides; Communities; Complex; Crystallization; Data; Data Collection; Databases; Detergents; Development; Disease; Engineering; Family; Goals; Hereditary Disease; Homology Modeling; Human; Integral Membrane Protein; Laboratories; Mammalian Cell; Mediating; Membrane Proteins; Methods; Molecular; Multi-Drug Resistance; Orthologous Gene; Pichia; Process; Property; Protein Structure Initiative; Proteins; Resolution; Resources; Roentgen Rays; Solubility; Structure; Synthetic Genes; System; Techniques; Technology; Therapeutic; Translations; Up-Regulation; base; biophysical properties; detector; experience; flexibility; instrumentation; member; neoplastic cell; protein folding; protein structure; screening; success; tool; web site

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.

事实上，每个分子穿过细胞膜的过程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至关重要，当这些过程受到干扰或破坏时，就会发生多种疾病，例如几种遗传性疾病或肿瘤细胞对多药耐药转运蛋白的上调。人类转运蛋白高分辨率结构的可用性对于定义其机制的分子结构基础至关重要。我们建议建立一个膜蛋白结构测定中心 TransportPDB，其目标是开发一种全面、有效的方法，用于研究目前在人类和 PSI 生物学中心的其他目标中发现的 48 个家族的 521 个转运蛋白的高分辨率 X 射线晶体结构。为此并实现 PSI 的目标，我们有以下具体目标： A1。我们将基于成熟的技术并利用我们成功结晶和解析完整膜蛋白的 X 射线结构的经验，建立高效的管道。该管道将​​基于几个关键原则：(a) 基于疾病相关性的目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) 独家使用真核表达系统（毕赤酵母和 293S 哺乳动物细胞），该系统已被证明可以提供适合结晶的功能性蛋白质，(c) 基于针对在两个表达系统中表达而优化的合成基因的构建体克隆，以及 (d) 使用最先进的数据收集技术进行适度衍射晶体。这种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直系同源物，从而朝着成功获得其 X 射线晶体结构的目标迈进。 A2。将开发高通量方法和技术，用于靶标的功能和生物物理表征，以快速识别维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人类转运蛋白晶体。将采用新的晶体安装方法，结合微束/光栅技术和提高数据收集灵敏度（皮拉图斯探测器），这对于适度衍射膜蛋白晶体可能具有决定性作用。 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。

项目成果

Directed evolution of P-glycoprotein cysteines reveals site-specific, non-conservative substitutions that preserve multidrug resistance.

P-糖蛋白半胱氨酸的定向进化揭示了保持多药耐药性的位点特异性、非保守性取代。

• DOI: 10.1042/bsr20140062
• 发表时间: 2014
• 期刊: Bioscience reports
• 影响因子: 4
• 作者: Swartz,DouglasJ;Mok,Leo;Botta,SriK;Singh,Anukriti;Altenberg,GuillermoA;Urbatsch,InaL
• 通讯作者: Urbatsch,InaL

A gene optimization strategy that enhances production of fully functional P-glycoprotein in Pichia pastoris.

• DOI: 10.1371/journal.pone.0022577
• 发表时间: 2011
• 期刊: PloS one
• 影响因子: 3.7
• 作者: Bai J;Swartz DJ;Protasevich II;Brouillette CG;Harrell PM;Hildebrandt E;Gasser B;Mattanovich D;Ward A;Chang G;Urbatsch IL
• 通讯作者: Urbatsch IL

P-glycoprotein is fully active after multiple tryptophan substitutions.

P-糖蛋白在多次色氨酸取代后具有完全活性。

• DOI: 10.1016/j.bbamem.2012.12.005
• 发表时间: 2013
• 期刊: Biochimica et biophysica acta
• 作者: Swartz,DouglasJ;Weber,Joachim;Urbatsch,InaL
• 通讯作者: Urbatsch,InaL