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Ciniawsky, Susanne (2015): Structural and functional insights into the mechanism of the Pex1/6 complex. Dissertation, LMU München: Faculty of Chemistry and Pharmacy



Peroxisomes are highly dynamic organelles of eukaryotic cells, carrying out essential oxidative metabolic processes. These organelles scavenge reactive oxygen species such as hydrogen peroxide (H2O2) and catabolise fatty acids, which are particular hallmarks and highly conserved features of peroxisomes among different species. Peroxisomal proteins and enzymes are encoded by nuclear DNA and therefore, targeted post-translationally into the peroxisomal matrix. A special class of proteins, collectively called peroxins, perform certain cellular tasks, such as peroxisomal matrix protein import or membrane development in order to maintain peroxisome biogenesis as well as a constant flux of matrix proteins into peroxisomes. The type II AAA+ peroxins Pex1/Pex6 are a core component of the peroxisomal matrix protein import system. ATPases of the AAA+ family of proteins generally assemble into large, macromolecular machines, structurally remodelling their substrate protein, which is driven by the hydrolysis of ATP. The main function of Pex1/6 complexes is to release the receptor Pex5 from peroxisomal membranes after matrix protein import. This relocation of Pex5 into the cytosol ensures a constant pool of available receptor molecules for subsequent cycles of protein import into peroxisomes. Accordingly, certain mutations in mammalian Pex1/Pex6 proteins compromise peroxisome biogenesis and thus, lipid metabolism, causing severe genetic Zellweger diseases in humans. In collaboration with Professor Ralf Erdmann and colleagues at the Ruhr-Universität Bochum, we characterize the structure and function of the AAA+ Pex1/6 complex from yeast Saccharomyces cerevisiae. Single particle electron microscopy (EM) in combination with biochemical assays allows us to analyze how ATP turnover is related to the biological function of the Pex1/6 complex. This study presents EM structures of Pex1/6 complexes assembled in the presence of ADP, ATP, ADP-AlFx and ATPγS, providing a comprehensive structural characterization of the heterohexameric type II AAA+ complex in different nucleotide states. Our EM reconstructions reveal an unexpected triangular overall shape, different than observed for the closely related and well-characterized homohexameric AAA+ protein p97. We show that the heterohexameric Pex1/6 complex is composed of a trimer of heterodimers with alternating subunit arrangement of Pex1 and Pex6 moieties. Furthermore, our results suggest that conserved aromatic residues, lining the central pore of the Pex1/6 D2 ring mediate substrate interactions. These residues correspond to substrate interaction regions in related AAA+ proteins. Comparing Pex1/6 EM reconstructions in different nucleotide states implicates that the mechanical function of Pex1/6 involves an N- to C-terminal protein translocation mechanism along the central pore. The Pex1/6 EM structures resolve symmetric and asymmetric large-scale domain motions, which likely create a power stroke during cycles of ATP binding and hydrolysis. We conclude that Pex5 is probably partially or completely unfolded while it is threaded through the central pore of Pex1/6 complexes. In addition, ATP hydrolysis assays of Pex1/Pex6 complexes containing single amino acid exchanges in individual Walker B motifs reveal that not all active sites are functionally equivalent. In isolated complexes, ATP turnover mainly occurs in Pex6 D2 domains, while Pex1 subunits sustain the structural integrity of the complex. We further resolve the structures of Pex1/6 Walker B variants and observe mutually exclusive protomer-protomer communication. In the Pex1/6 complex, a Walker B mutation induces ATP hydrolysis in the adjacent D2 domain, presenting a structural framework of protomer-protomer communication in the AAA+ heterohexamer.