The structure and oxidation of the eye lens chaperone αA-crystallin.

The small heat shock protein αA-crystallin is a molecular chaperone important for the optical properties of the vertebrate eye lens. It forms heterogeneous oligomeric ensembles. We determined the structures of human αA-crystallin oligomers by combining cryo-electron microscopy, cross-linking/mass spectrometry, NMR spectroscopy and molecular modeling. The different oligomers can be interconverted by the addition or subtraction of tetramers, leading to mainly 12-, 16- and 20-meric assemblies in which interactions between N-terminal regions are important. Cross-dimer domain-swapping of the C-terminal region is a determinant of αA-crystallin heterogeneity. Human αA-crystallin contains two cysteines, which can form an intramolecular disulfide in vivo. Oxidation in vitro requires conformational changes and oligomer dissociation. The oxidized oligomers, which are larger than reduced αA-crystallin and destabilized against unfolding, are active chaperones and can transfer the disulfide to destabilized substrate proteins. The insight into the structure and function of αA-crystallin provides a basis for understanding its role in the eye lens.

Diversity of COP9 signalosome structures and functional consequences.

The COP9 signalosome (CSN) is a regulator of the ubiquitin (Ub) proteasome system (UPS). It interacts with hundreds of cullin-RING ubiquitin E3 ligases (CRLs) and regulates their activity by removing the Ub-like protein Nedd8 from cullins. In mammalian cells 7 different cullins exist which form CRLs with adaptor proteins and with a large number of substrate recognition subunits such as F-box and BTB proteins. This large variety of CRL-complexes is deneddylated by the CSN. The capacity of the CSN to interact with numerous types of CRL complexes can be explained by its structural diversity, which allows different CSN variants to interact with different binding partners and substrates and enables different subunit expression profiles. Diversity of CSN complexes presumably occurs by: (1) flexibility of CSN holo complex structure; (2) formation of CSN mini complexes and free CSN subunits and (3) generation of CSN variants via integration of CSN subunit isoforms. In this review we will discuss the structural diversity of the CSN complex and possible functional consequences.

Electron microscopy and in vitro deneddylation reveal similar architectures and biochemistry of isolated human and Flag-mouse COP9 signalosome complexes.

The COP9 signalosome (CSN) is a regulator of the ubiquitin (Ub) proteasome system (UPS). In the UPS, proteins are Ub-labeled for degradation by Ub ligases conferring substrate specificity. The CSN controls a large family of Ub ligases called cullin-RING ligases (CRLs), which ubiquitinate cell cycle regulators, transcription factors and DNA damage response proteins. The CSN possesses structural similarities with the 26S proteasome Lid complex and the translation initiation complex 3 (eIF3) indicating similar ancestry and function. Initial structures were obtained 14years ago by 2D electron microscopy (EM). Recently, first 3D molecular models of the CSN were created on the basis of negative-stain EM and single-particle analysis, mostly with recombinant complexes. Here, we compare deneddylating activity and structural features of CSN complexes purified in an elaborate procedure from human erythrocytes and efficiently pulled down from mouse Flag-CSN2 B8 fibroblasts. In an in vitro deneddylation assay both the human and the mouse CSN complexes deneddylated Nedd8-Cul1 with comparable rates. 3D structural models of the erythrocyte CSN as well as of the mouse Flag-CSN were generated by negative stain EM and by cryo-EM. Both complexes show a central U-shaped segment from which several arms emanate. This structure, called the horseshoe, is formed by the PCI domain subunits. CSN5 and CSN6 point away from the horseshoe. Compared to 3D models of negatively stained CSN complexes, densities assigned to CSN2 and CSN4 are better defined in the cryo-map. Because biochemical and structural results obtained with CSN complexes isolated from human erythrocytes and purified by Flag-CSN pulldown from mouse B8 fibroblasts are very similar, Flag-CSN pulldowns are a proper alternative to CSN preparation from erythrocytes.

The structure of human tripeptidyl peptidase II as determined by a hybrid approach.

Tripeptidyl-peptidase II (TPPII) is a high molecular mass (∼5 MDa) serine protease, which is thought to act downstream of the 26S proteasome, cleaving peptides released by the latter. Here, the structure of human TPPII (HsTPPII) has been determined to subnanometer resolution by cryoelectron microscopy and single-particle analysis. The complex is built from two strands forming a quasihelical structure harboring a complex system of inner cavities. HsTPPII particles exhibit some polymorphism resulting in complexes consisting of nine or of eight dimers per strand. To obtain deeper insights into the architecture and function of HsTPPII, we have created a pseudoatomic structure of the HsTPPII spindle using a comparative model of HsTPPII dimers and molecular dynamics flexible fitting. Analyses of the resulting hybrid structure of the HsTPPII holocomplex provide new insights into the mechanism of maturation and activation.

Structure and function of tripeptidyl peptidase II, a giant cytosolic protease.

Tripeptidyl peptidase II is the largest known eukaryotic peptidase. It has been described as a multi-purpose peptidase, which, in addition to its house-keeping function in intracellular protein degradation, plays a role in several vital cellular processes such as antigen processing, apoptosis, or cell division, and is involved in diseases like muscle wasting, obesity, and in cancer. Biochemical studies and bioinformatics have identified TPPII as a subtilase, but its structure is very unusual: it forms a large homooligomeric complex (6 MDa) with a spindle-like shape. Recently, the high-resolution structure of TPPII homodimers (300 kDa) was solved and a hybrid structure of the holocomplex built of 20 dimers was obtained by docking it into the EM-density. Here, we summarize our current knowledge about TPPII with a focus on structural aspects. This article is part of a Special Issue entitled: Proteolysis 50 years after the discovery of lysosome.

Molecular ruler of tripeptidylpeptidase II: mechanistic principle of exopeptidase selectivity.

The structure of tripeptidylpeptidase II (TPPII) has shown that it belongs to the group of exopeptidases which use a double-Glu motif to convey aminopeptidase activity. TPPII has been implicated in vital biological processes. At least one of these, antigen processing, requires the involvement of its endopeptidase activity. In order to understand the extent and molecular basis of this unusual functional promiscuity we have performed a systematic kinetic analysis of wild type Drosophila melanogaster TPPII and five point mutants of the double-Glu-motif (E312/E343) involving natural substrates. Unlike the known double-Glu motives of other exopeptidases, the double-Glu motif of TPPII is distinctly asymmetrical: E312 is the crucial determinant of the aminotripeptidolytic ruler mechanism. It both blocks the active-site cleft at substrate position P4 and forms a salt bridge with the N-terminus of the substrate. In contrast, E343 forms a much weaker salt bridge than E312 and it does not have a blocking role. An endopeptidase substrate can bind at relatively high affinity if the length of the substrate permits binding to several S' sites. However, the lacking alignment of the substrate by the double-Glu motif causes the endopeptidolytic K(cat)/K(M) of TPPII to be very low.

Hybrid molecular structure of the giant protease tripeptidyl peptidase II.

Tripeptidyl peptidase II (TPP II) is the largest known eukaryotic protease (6 MDa). It is believed to act downstream of the 26S proteasome, cleaving tripeptides from the N termini of longer peptides, and it is implicated in numerous cellular processes. Here we report the structure of Drosophila TPP II determined by a hybrid approach. We solved the structure of the dimer by X-ray crystallography and docked it into the three-dimensional map of the holocomplex, which we obtained by single-particle cryo-electron microscopy. The resulting structure reveals the compartmentalization of the active sites inside a system of chambers and suggests the existence of a molecular ruler determining the size of the cleavage products. Furthermore, the structure suggests a model for activation of TPP II involving the relocation of a flexible loop and a repositioning of the active-site serine, coupling it to holocomplex assembly and active-site sequestration.

Two unique membrane-bound AAA proteins from Sulfolobus solfataricus.

Thermoacidophilic crenarchaea of the genus Sulfolobus contain six AAA (ATPase associated with various cellular activities) proteins, including a proteasome-associated ATPase, a Vps4 (vacuolar protein sorting 4) homologue, and two Cdc48 (cell-division cycle 48)-like proteins. The last two AAA proteins are deeply branching divergent members of this family without close relatives outside the Sulfolobales. Both proteins have two nucleotide-binding domains and, unlike other members of the family, they seem to lack folded N-terminal domains. Instead, they contain N-terminal extensions of approx. 50 residues, which are predicted to be unstructured, except for a single transmembrane helix. We have analysed the two proteins, MBA (membrane-bound AAA) 1 and MBA2, by computational and experimental means. They appear to be monophyletic and to share a common ancestor with the Cdc48 clade. Both are membrane-bound and active as nucleotidases upon heterologous expression in Escherichia coli. They form ring complexes, which are stable after solubilization in a mild detergent and whose formation is dependent on the presence of the N-terminal extensions.

Visualizing cells at the nanoscale.

Cryogenic electron tomography (cryo- ET) enables the 3D visualization of biological material at a previously unseeable scale. Carefully controlled cryogenic specimen preparation avoids the artefacts that are notorious to conventional electron microscopy specimen preparation. To date, studies employing cryo- ET have mostly been restricted to isolated macromolecular assemblies, small prokaryotic cells or thin regions of eukaryotic cells owing to the limited penetration depth of electrons through ice-embedded preparations. Recent progress in cryosectioning makes it possible to acquire tomograms from many kinds of vitrified cells and tissues. The systematic and comprehensive interpretation of such tomograms will provide unprecedented insight into the molecular organization of cellular landscapes.

Size matters for the tripeptidylpeptidase II complex from Drosophila: The 6-MDa spindle form stabilizes the activated state.

Tripeptidylpeptidase II (TPP II) is an exopeptidase of the subtilisin type of serine proteases, a key component of the protein degradation cascade in many eukaryotes, which cleaves tripeptides from the N terminus of proteasome-released products. The Drosophila TPP II is a large homooligomeric complex (approximately 6 MDa) that is organized in a unique repetitive structure with two strands each composed of ten stacked homodimers; two strands intertwine to form a spindle-shaped structure. We report a novel procedure of preparing an active, structurally homogeneous TPP II holo-complex overexpressed in Escherichia coli. Assembly studies revealed that the specific activity of TPP II increases with oligomer size, which in turn is strongly concentration-dependent. At a TPP II concentration such as prevailing in Drosophila, equilibration of size and activity proceeds on a time scale of hours and leads to spindle formation at a TPP II concentration of > or =0.03 mg/ml. Before equilibrium is reached, activation lags behind assembly, suggesting that activation occurs in a two-step process consisting of (i) assembly and (ii) a subsequent conformational change leading to a switch from basal to full activity. We propose a model consistent with the hyperbolic increase of activity with oligomer size. Spindle formation by strand pairing causes both significant thermodynamic and kinetic stabilization. The strands inherently heterogeneous in length are thus locked into a discrete oligomeric state. Our data indicate that the unique spindle form of the holo-complex represents an assembly motif stabilizing a highly active state.

Characterization of AMA, a new AAA protein from Archaeoglobus and methanogenic archaea.

We have previously reported a new group of AAA proteins, which is only found in Archaeoglobus and methanogenic archaea (AMA). The proteins are phylogenetically basal to the metalloprotease clade and their N-terminal domain is homologous to the beta-clam part of the N-domain of CDC48-like proteins. Here we report the biochemical and biophysical characterization of Archaeoglobus fulgidus AMA, and of its isolated N-terminal (AMA-N) and ATPase (AMA-DeltaN) domains. AfAMA forms hexameric complexes, as does AMA-N, while AMA-DeltaN only forms dimers. The ability to hexamerize is dependent on the integrity of a GYPL motif in AMA-N, which resembles the pore motif of FtsH and HslU. While the physiological function of AMA is unknown, we show that it has ATP-dependent chaperone activity and can prevent the thermal aggregation of proteins in vitro. The ability to interact with non-native proteins resides in the N-domain and is energy-independent.

20S proteasomes have the potential to keep substrates in store for continual degradation.

The 20S core of the proteasome, which together with the regulatory particle plays a major role in the degradation of proteins in eukaryotic cells, is traversed by an internal system of cavities, namely two antechambers and one central proteolytic chamber. Little is known about the mechanisms underlying substrate binding and translocation of polypeptide chains into the interior of 20S proteasomes. Specifically, the role of the antechambers is not fully understood, and the number of substrate molecules sequestered within the internal cavities at any one time is unknown. Here we have shown that by applying both electron microscopy and tandem mass spectrometry (MS) approaches to this multisubunit complex we obtain precise information regarding the stoichiometry and location of substrates within the three chambers. The dissociation pattern in tandem MS allows us to conclude that a maximum of three green fluorescent protein and four cytochrome c substrate molecules are bound within the cavities. Our results also show that >95% of the population of proteasome molecules contain the maximum number of partially folded substrates. Moreover, we deduce that one green fluorescent protein or two cytochrome c molecules must reside within the central proteolytic chamber while the remaining substrate molecules occupy, singly, both antechambers. The results imply therefore an additional role for 20S proteasomes in the storage of substrates prior to their degradation, specifically in cases where translocation rates are slower than proteolysis. More generally, the ability to locate relatively small protein ligands sequestered within the 28-subunit core particle highlights the tremendous potential of tandem MS for deciphering substrate binding within large macromolecular assemblies.

Particle picking by segmentation: a comparative study with SPIDER-based manual particle picking.

Boxing hundreds of thousands of particles in low-dose electron micrographs is one of the major bottle-necks in advancing toward achieving atomic resolution reconstructions of biological macromolecules. We have shown that a combination of pre-processing operations and segmentation can be used as an effective, automatic tool for identifying and boxing single-particle images. This paper provides a brief description of how this method has been applied to a large data set of micrographs of ice-embedded ribosomes, including a comparative analysis of the efficiency of the method. Some results on processing micrographs of tripeptidyl peptidase II particles are also shown. In both cases, we have achieved our goal of selecting at least 80% of the particles that an expert would select with less than 10% false positives.

VAT, the thermoplasma homolog of mammalian p97/VCP, is an N domain-regulated protein unfoldase.

The Thermoplasma VCP-like ATPase from Thermoplasma acidophilum (VAT) ATPase is a member of the two-domain AAA ATPases and homologous to the mammalian p97/VCP and NSF proteins. We show here that the VAT ATPase complex unfolds green fluorescent protein (GFP) labeled with the ssrA-degradation tag. Increasing the Mg2+ concentration derepresses the ATPase activity and concomitantly stimulates the unfolding activity of VAT. Similarly, the VATDeltaN complex, a mutant of VAT deleted for the N domain, displays up to 24-fold enhanced ATP hydrolysis and 250-fold enhanced GFP unfolding activity when compared with wild-type VAT. To determine the individual contribution of the two AAA domains to ATP hydrolysis and GFP unfolding we performed extensive site-directed mutagenesis of the Walker A, Walker B, sensor-1, and pore residues in both AAA domains. Analysis of the VAT mutant proteins, where ATP hydrolysis was confined to a single AAA domain, revealed that the first domain (D1) is sufficient to exert GFP unfolding indistinguishable from wild-type VAT, while the second AAA domain (D2), although active, is significantly less efficient than wild-type VAT. A single conserved aromatic residue in the D1 section of the pore was found to be essential for GFP unfolding. In contrast, two neighboring residues in the D2 section of the pore had to be exchanged simultaneously, to achieve a drastic inhibition of GFP unfolding.

Molecular architecture and assembly mechanism of Drosophila tripeptidyl peptidase II.

In eukaryotes, tripeptidyl peptidase II (TPPII) is a crucial component of the proteolytic cascade acting downstream of the 26S proteasome in the ubiquitin-proteasome pathway. It is an amino peptidase belonging to the subtilase family removing tripeptides from the free N terminus of oligopeptides. The 150-kDa subunits of Drosophila TPPII assemble into a giant proteolytic complex of 6 MDa with a remarkable architecture consisting of two segmented and twisted strands that form a spindle-shaped structure. A refined 3D model has been obtained by cryoelectron microscopy, which reveals details of the molecular architecture and, in conjunction with biochemical data, provides insight into the assembly mechanism. The building blocks of this complex are apparently dimers, within which the 150-kDa monomers are oriented head to head. Stacking of these dimers leads to the formation of twisted single strands, two of which comprise the fully assembled spindle. This spindle also forms when TPPII is heterologously expressed in Escherichia coli, demonstrating that no scaffolding protein is required for complex formation and length determination. Reciprocal interactions of the N-terminal part of subunits from neighboring strands are probably involved in the formation of the native quaternary structure, lending the TPPII spindle a stability higher than that of single strands.

Lignin distribution in wood cell walls determined by TEM and backscattered SEM techniques.

The lignin distribution in cell walls of spruce and beech wood was determined by high-voltage transmission-electron-microscopy (TEM) in sections stained with potassium permanganate as well as by field-emission-scanning-electron-microscopy (FE-SEM) combined with a back-scattered electron detector on mercurized specimens. The latter is a new technique based on the mercurization of lignin and the concomitant visualization of mercury by back-scattered electron microscopy (BSE). Due to this combination it was possible to obtain a visualized overview of the lignin distribution across the different layers of the cell wall. To our knowledge, this combined method was used the first time to analyse the lignin distribution in cell walls. In agreement with previous work the highest lignin levels were found in the compound middle lamella and the cell corners. Back-scattered FE-SEM allows the lignin distribution in the pit membrane of bordered pits as well as in the various cell wall layers to be shown. In addition, by using TEM as well as SEM we observed that lignin closely follows the cellulose microfibril orientation in the secondary cell wall. From these observations, we conclude that the polymerisation of monolignols is affected by the arrangement of the polysaccharides which constitute the cell wall.

A giant protease with a twist: the TPP II complex from Drosophila studied by electron microscopy.

Tripeptidyl peptidase II (TPP II) is an exopeptidase of the subtilisin type of serine proteases that is thought to act downstream of the proteasome in the ubiquitin-proteasome pathway. Recently, a key role in a pathway parallel to the ubiquitin-proteasome pathway has been ascribed to TPP II, which forms a giant protease complex in mammalian cells. Here, we report the 900-fold purification of TPP II from Drosophila eggs and demonstrate via cryo-electron microscopy that TPP II from Drosophila melanogaster also forms a giant protease complex. The presented three-dimensional reconstruction of the 57 x 27 nm TPP II complex at 3.3 nm resolution reveals that the 150 kDa subunits form a superstructure composed of two segmented and twisted strands. Each strand is 12.5 nm in width and composed of 11 segments that enclose a central channel.

Electron cryo-microscopy of VAT, the archaeal p97/CDC48 homologue from Thermoplasma acidophilum.

VAT (valosine containing protein-like ATPase from Thermoplasma acidophilum), an archaeal member of the AAA-family (ATPases associated with a variety of cellular activities) that possesses foldase as well as unfoldase-activity, forms homo-hexameric rings like its eukaryotic homologues p97 and CDC48. The VAT-monomer exhibits the tripartite domain architecture typical for type II AAA-ATPases: N-D1-D2, whereby N is the substrate binding N-terminal domain preceding domains D1 and D2, both containing AAA-modules. Recent 3-D reconstructions of VAT and p97 as obtained by electron microscopy suffer from weakly represented N-domains, probably a consequence of their flexible linkage to the hexameric core. Here we used electron cryo-microscopy and 3-D reconstruction of single particles in order to generate a 3-D model of VAT at 2.3 nm resolution. The hexameric core of the VAT-complex (diameter 13.2 nm, height 8.4 nm) encloses a central cavity and the substrate-binding N-domains are clearly arranged in the upper periphery. Comparison with the p97 3-D reconstruction and the recently determined crystal structure of p97-N-D1 suggests a tail-to-tail arrangement of D1 and D2 in VAT.