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

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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.