Structural biology of the translocation pore of the peroxisomal protein import machinery

过氧化物酶体蛋白输入机制易位孔的结构生物学

• 批准号: 237561355
• 负责人: Professor Dr. Michael Sattler
• 金额: --
• 依托单位: Lehrstuhl für Biomolekulare NMR-Spektroskopie
• 依托单位国家: 德国
• 项目类别: Research Units
• 财政年份: 2013
• 资助国家: 德国
• 起止时间: 2012-12-31 至 2021-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/237561355?language=en
• 关键词: Structural; biology; translocation; pore; peroxisomal

Structural elucidation of the peroxisomal pore poses a major remaining challenge in life sciences, as the pore is non-permanent and behaves highly dynamically during each translocation cycle. Within the overarching vision of the entire PerTrans network, a common aim of the two project groups Sattler and Wilmanns is to synergistically capture the high-resolution structures of single components and sub-complexes of known translocons.In the first funding period, the Wilmanns group has structurally and functionally unraveled how peroxisomal cargo recognition is regulated by the ability for major conformational changes of the Pex5p receptor. In addition, the group has engaged in the production and biophysical characterization of translocon docking complexes and a largely uncharacterized release factor Pex8p. Solution studies using NMR and SAXS by the Sattler group have provided structural insight into novel, unexpected protein interactions involving key factors of the peroxisomal translocon and highlighted the role of intrinsically disordered regions, which are predominant in the N-terminal halves of the human PEX5 receptor and the PMP transport factor PEX19.To provide a mechanistic understanding of the peroxisomal translocon the two groups of this project (Wilmans, Sattler) will further develop and employ an integrated structural biology approach combining crystallography, NMR-spectroscopy, small angle X-ray and neutron scattering, and utilize cryo-electron microscopy and cross-linking/mass spectrometry with the partners of Projects 2 and 6, respectively. We will pursue two principal aims to study the peroxisomal translocon as a holo complex and consider the role of large instrinsically disordered regions in its key components. In Aim 1 the Wilmanns lab will focus on structural work of four sub-projects involved in cargo recognition (Pex5p-Pcs60p, Pex9p), cargo release (Pex8p), and cargo translocation by focusing on the PTS2 pore (in collaboration with Project 2). In Aim 2 the Sattler group will characterize the conformation and dynamics of full-length PEX5, PEX14, and PEX13 proteins and their reconstituted complexes alone and in the presence of cargo proteins. The results of Project 3 are expected to provide a comprehensive picture of the conformation and dynamics of the key components of the peroxisomal translocon and will provide a basis to facilitate determining the architecture of the entire peroxisomal translocon within the PerTans consortium.

过氧化物酶体孔的结构解析在生命科学中构成了一个主要的剩余挑战，因为孔是非永久性的，并且在每个易位周期期间表现出高度动态性。在整个PerTrans网络的总体愿景中，Sattler和Wilmanns两个项目组的共同目标是协同捕获已知translocons的单个组分和子复合物的高分辨率结构。在第一个资助期内，Wilmanns小组在结构和功能上揭示了过氧化物酶体货物识别如何受到Pex 5 p受体主要构象变化能力的调节。此外，该小组还参与了translocon对接复合物和一种基本上未表征的释放因子Pex 8 p的生产和生物物理表征。Sattler小组使用NMR和SAXS进行的溶液研究为涉及过氧化物酶体转位子关键因子的新型、意想不到的蛋白质相互作用提供了结构见解，并强调了固有无序区域的作用，在人PEX 5受体和PMP转运因子PEX 19的N-末端占优势。为了提供对过氧化物酶体转位子的机制的理解，本项目的两个研究组（Wilmans，Sattler）将进一步开发和采用结合晶体学，NMR光谱学，小角X射线和中子散射的综合结构生物学方法，并分别与项目2和6的合作伙伴利用低温电子显微镜和交联/质谱法。我们将追求两个主要目标，研究过氧化物酶体的translocon作为一个全息复合物，并考虑在其关键组成部分的大的电子自旋无序区域的作用。在目标1中，Wilmanns实验室将重点关注四个子项目的结构工作，这些子项目涉及货物识别（Pex 5 p-Pcs 60 p，Pex 9 p），货物释放（Pex 8 p）和货物易位，重点关注PTS 2孔（与项目2合作）。在目标2中，Sattler小组将表征全长PEX 5、PEX 14和PEX 13蛋白及其单独和在货物蛋白存在下的重构复合物的构象和动力学。预计项目3的结果将提供过氧化物酶体易位子的关键组分的构象和动力学的全面图片，并将提供一个基础，以促进确定整个过氧化物酶体易位子的PerTans财团内的架构。