Structural and functional insights into the mechanism of the Pex1/6 complex

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

Structural and functional insights into the mechanism of the Pex1/6 complex

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

Molecular snapshots of the Pex1/6 AAA + complex in action

The peroxisomal proteins Pex1 and Pex6 form a heterohexameric type II AAA+ ATPase complex, which fuels essential protein transport across peroxisomal membranes. Mutations in either ATPase in humans can lead to severe peroxisomal disorders and early death. We present an extensive structural and biochemical analysis of the yeast Pex1/6 complex. The heterohexamer forms a trimer of Pex1/6 dimers with a triangular geometry that is atypical for AAA+ complexes. While the C-terminal nucleotide-binding domains (D2) of Pex6 constitute the main ATPase activity of the complex, both D2 harbour essential sub-strate-binding motifs. ATP hydrolysis results in a pumping motion of the complex, suggesting that Pex1/6 function involves substrate translocation through its central channel. Mutation of the Walker B motif in one D2 domain leads to ATP hydrolysis in the neighbouring domain, giving structural insights into inter-domain communication of these unique heterohexameric AAA + assemblies

Peroxisome biogenesis, protein targeting mechanisms and PEX gene functions in plants

Peroxisomes play diverse and important roles in plants. The functions of peroxisomes are dependent upon their steady state protein composition which in turn reflects the balance of formation and turnover of the organelle. Protein import and turnover of constituent peroxisomal proteins is controlled by the state of cell growth and environment. The evolutionary origin of the peroxisome and the role of the endoplasmic reticulum in peroxisome biogenesis is discussed, as informed by studies of the trafficking of peroxisome membrane proteins. The process of matrix protein import in plants and its similarities and differences with peroxisomes in other organisms is presented and discussed in the context of peroxin distribution across the green plants

Covalent Label Transfer Between Peroxisomal Importomer Components Reveals Export-Driven Import Interactions

Peroxisomes are vital metabolic organelles found in almost all eukaryotic organisms, and they rely exclusively on import of their matrix protein content from the cytosol. In vitro import of proteins into isolated peroxisomal fractions has provided a wealth of knowledge on the import process. However, the common method of protease protection garnered no information on the import of an N-terminally truncated PEX5 (PEX5C) receptor construct or peroxisomal Malate Dehydrogenase 1 (pMDH1) cargo protein into sunflower peroxisomes, owing to high degrees of protease susceptibility or resistance, respectively. Here, we present a means for analysis of in vitro import through a covalent biotin label transfer, and employ this method to the import of PEX5C. Label transfer demonstrates that PEX5C construct is monomeric in the conditions of the import assay. This technique was capable of identifying the PEX5-PEX14 interaction as the first interaction of the import process through competition experiments. Labelling of the peroxisomal protein import machinery by PEX5C demonstrated that this interaction was independent of added cargo protein, and strikingly, the interaction between PEX5C and the import machinery was shown to be ATP-dependent. These important mechanistic insights highlight the power of label transfer in studying interactions, rather than proteins, of interest, and demonstrate that this technique should be applied to future studies of peroxisomal in vitro import

Peroxisome protein import: a complex journey.

The import of proteins into peroxisomes possesses many unusual features such as the ability to import folded proteins, and a surprising diversity of targeting signals with differing affinities that can be recognised by the same receptor. As understanding of the structure and function of many components of the protein import machinery has grown, an increasingly complex network of factors affecting each step of the import pathway has emerged. Structural studies have revealed the presence of additional interactions between cargo proteins and the PEX5 receptor that affect import potential, with a subtle network of cargo-induced conformational changes in PEX5 being involved in the import process. Biochemical studies have also indicated an interdependence of receptor-cargo import with release of unloaded receptor from the peroxisome. Here we provide an update on recent literature concerning mechanisms of protein import into peroxisomes

Infrared spectroelectrochemical studies of coordination compounds.

The discovery of metal-containing constituents in many biological systems has stimulated research into the redox activity of transition metal complexes with non-innocent ligands. Interest has centred upon synthesising model compounds, which have similar spectroscopic or structural characteristics to the metal site of metalloproteins. In this way, a better understanding is gained of the behaviour of metalloenzymes such as the molybdenum-containing co-factors of nitrogenase, the oxotransferases, and the iron-containing ferredoxin electron-transfer agents. The work carried out involved the electrochemical study of inorganic species, supplemented by infrared and ultra-violet/visible spectroelectrochemical experiments. By these means, it was possible to obtain a more precise evaluation of the changes occurring on oxidation or reduction, this being related to the frontier molecular orbitals of the complex. The extremely reactive and short-lived nature of many electrogenerated products required the development of controlled, strictly anaerobic sample-handling procedures for spectroelectrochemical experiments. This was achieved by modifying the design of the spectroelectrochemical cell. The limitations of standard spectroelectrochemical techniques for the study of reactive short-lived species in solution prompted the development of the "Modulation Technique". This involved computer control and synchronisation of spectral data acquisition with the change of potential applied to the electrode. By the reversible generation, for a maximum of 2-3s, of the reactive species reliable spectroscopic results could be obtained. Modification of the working electrode enabled these experiments to be performed on a reduced timescale and at reduced temperatures to those previously possible. Using this technique, spectra were recorded of a large range of previously unobserved and unstable species, such as the tetra-anionic tris maleonitriledithiolate complexes of vanadium, chromium, molybdenum and rhenium. The complicated series of chemical and electrochemical transformations initiated by oxidation of the oxomolybdenum compound MoOmnt22- was also investigated and the intermediates identified. Significant insights were gained into the behaviour upon reduction of the carbonyl- and nitrosyl-containing compound cpMo(CO)2(NO). Spectral data were obtained for Fe4S4(NO)4, a model compound for the iron-sulphur electron transfer proteins, in four different oxidation states, the evidence suggesting that the molecule retains its stable, cubane-like structure throughout all the redox steps

Mechanism of Enzyme Repair by the AAA(+) Chaperone Rubisco Activase

How AAA(+) chaperones conformationally remodel specific target proteins in an ATP-dependent manner is not well understood. Here, we investigated the mechanism of the AAA(+) protein Rubisco activase (Rca) in metabolic repair of the photosynthetic enzyme Rubisco, a complex of eight large (RbcL) and eight small (RbcS) subunits containing eight catalytic sites. Rubisco is prone to inhibition by tight-binding sugar phosphates, whose removal is catalyzed by Rca. We engineered a stable Rca hexamer ring and analyzed its functional interaction with Rubisco. Hydrogen/deuterium exchange and chemical crosslinking showed that Rca structurally destabilizes elements of the Rubisco active site with remarkable selectivity. Cryo-electron microscopy revealed that Rca docks onto Rubisco over one active site at a time, positioning the C-terminal strand of RbcL, which stabilizes the catalytic center, for access to the Rca hexamer pore. The pulling force of Rca is fine-tuned to avoid global destabilization and allow for precise enzyme repair