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

对过氧化体毛孔的结构解释是生命科学中的一个主要剩余挑战，因为该毛孔是非永久性的，在每个易位周期中都具有高度的动态行为。在整个Pertrans网络的总体愿景中，Sattler和Wilmanns两个项目组的一个共同目标是协同捕获已知转运蛋白的单个组分和亚复合体的高分辨率结构。在第一个资助期，Wilmanns小组从结构和功能上揭示了Pex5p受体的主要构象变化如何调节过氧化物体货物识别。此外，该小组还参与了转运子对接复合体的生产和生物物理表征，以及一种基本上未被表征的释放因子Pex8p。Sattler小组使用核磁共振和SAXS进行的解决方案研究提供了对涉及过氧体转位关键因素的新颖的、意想不到的蛋白质相互作用的结构洞察，并强调了内在无序区域的作用，这些区域主要位于人类PEX5受体和PMP运输因子PEX19的N-末端半部分。为了提供对过氧体转位的机制理解，该项目的两组(Wilmann，Sattler)将进一步开发和使用结合结晶学、核磁共振光谱、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/质谱仪。我们将追求两个主要目标，将过氧体转位蛋白作为一个全息复合体来研究，并考虑大的无序区域在其关键成分中的作用。在目标1中，Wilmanns实验室将侧重于涉及货物识别(Pex5p-Pcs60p、Pex9p)、货物释放(Pex8p)和货物转运的四个子项目的结构工作(与项目2合作)。在目标2中，Sattler小组将表征全长PEX5、PEX14和PEX13蛋白及其重组复合体的构象和动力学，以及在货运蛋白存在下的构象和动力学。预计项目3的结果将提供过氧体转位子关键成分的构象和动力学的全面图景，并将为确定PerTans联合体内整个过氧体转位子的结构提供基础。

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.