Structural Characterization of the TOC Protein Translocon Machinery

TOC 蛋白易位机的结构表征

• 批准号: 9900017
• 负责人: Nicholas Noinaj
• 金额: $ 31.58万
• 依托单位: PURDUE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-05-01 至 2023-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9900017
• 关键词: Antibiotics; Apicomplexa; Artificial Membranes; Biological Assay; Biological Models; Biology; Cause of Death; Chloroplasts; Communicable Diseases; Complex; Country; Cryoelectron Microscopy; Cytoplasm; Defect; Destinations; Development; Drug usage; Energy Supply; Falciparum Malaria; Genes; Gram-Negative Bacteria; Guanosine Triphosphate Phosphohydrolases; Homology Modeling; Human; In Vitro; Infection; Malaria; Mediating; Membrane; Mitochondria; Modeling; Molecular; Molecular Chaperones; Molecular Machines; Mouse-ear Cress; Multi-Drug Resistance; Mutagenesis; N-terminal; Neurologic; Nuclear; Organelles; Parasites; Plasmodium falciparum; Plastids; Protein Export Pathway; Protein Import; Protein Precursors; Proteins; Reporting; Research; Resolution; Roentgen Rays; Role; Structure; System; Therapeutic; Toxoplasma; Toxoplasma gondii; Toxoplasmosis; Vaccines; X-Ray Crystallography; base; biophysical techniques; improved; nanodisk; novel; pathogen; polypeptide; protein transport; proteoliposomes; reconstitution; reconstruction; resistant strain

Project Summary Infectious diseases cause widespread sickness throughout the world each year and are the second leading cause of death, particularly in underdeveloped countries. And with the emergence of multi-drug resistance strains, the necessity for new, more effective, and more sustainable therapies is immediate. Included in these infectious diseases are the apicomplexa which includes Toxoplasma gondii and Plasmodium falciparum, the parasites causing toxoplasmosis and malaria, respectively. These parasites contain a unique plastid-like organelle called an apicoplast which contains four membranes and therefore have evolved a complex system for importing and exporting proteins across these membranes. These essential import/export machineries are ideal targets for novel antibiotics against these pathogens. Many of these translocon machineries are also conserved in other higher eukaryotic organelles such as chloroplasts and mitochondria, where a large majority of genes are nuclear encoded and therefore must be imported post-translationally. One such machinery is the conserved translocon of the outer membrane in chloroplasts (TOC) complex from Arabidopsis thaliana, a model system for studying chloroplast biology. The TOC complex consists of primarily three components, Toc33/34 and Toc159, both GTPases containing an N-terminal transmembrane helix anchoring them into the outer membrane, and Toc75, a 16- stranded β-barrel membrane-spanning translocon. While mechanistic models have been put forth for how the TOC complex functions, they have remained largely unproven due to the lack of structural characterization, which is needed to stitch together all the pieces of the mechanistic puzzle. In our studies, we will use biophysical methods, X-ray crystallography, cryo-electron microscopy, and small-angle X-ray scattering to structurally and functionally characterize this specialized machinery. Our results will fill a longstanding gap in the field and will be essential for piecing together the mechanism for how the TOC complex functions in protein import in apicomplexa and chloroplasts.

项目概要 传染病每年在全世界造成广泛的疾病，是第二大疾病 死亡的主要原因，特别是在不发达国家。 由于耐药菌株的存在，迫切需要新的、更有效、更可持续的疗法。 这些传染病包括尖复合体，其中包括弓形虫和 恶性疟原虫，分别引起弓形虫病和疟疾的寄生虫。 含有一种独特的类质体细胞器，称为顶端质体，它包含四个膜，因此 已经进化出一个复杂的系统，用于跨这些膜输入和输出蛋白质。 重要的进出口机械是针对这些病原体的新型抗生素的理想目标。 这些易位子机器也保存在其他高等真核细胞器中，例如 叶绿体和线粒体，其中大部分基因是核编码的，因此必须 翻译后输入的一种机制是外膜的保守易位子。 来自拟南芥的叶绿体 (TOC) 复合物，这是研究叶绿体生物学的模型系统。 TOC 复合物主要由三个组分组成：Toc33/34 和 Toc159，均为 GTPases 包含将它们锚定到外膜的 N 端跨膜螺旋，以及 Toc75（一种 16- 搁浅的β-桶跨膜易位子已经提出了机制模型。 TOC 复杂的功能，由于缺乏结构性，它们在很大程度上仍未得到证实。 在我们的研究中，需要将机械拼图的所有部分拼凑在一起。 我们将使用生物物理方法、X射线晶体学、冷冻电子显微镜和小角度X射线 我们的结果将填补这一特殊机械的结构和功能特征。 该领域长期存在的差距，对于整合 TOC 的机制至关重要 顶端复合体和叶绿体中蛋白质输入的复杂功能。