The microtubule-associated protein, tau, is the major subunit of neurofibrillary tangles associated with neurodegenerative conditions, such as Alzheimer's disease. In the cell, however, tau aggregation can be prevented by a class of proteins known as molecular chaperones. While numerous chaperones are known to interact with tau, though, little is known regarding the mechanisms by which these prevent tau aggregation. Here, we describe the effects of ATP-independent Hsp40 chaperones, DNAJA2 and DNAJB1, on tau amyloid-fiber formation, and compare these to the small heat-shock protein HSPB1. We find that the chaperones play complementary roles, with each preventing tau aggregation differently and interacting with distinct sets of tau species. Whereas HSPB1 only binds tau monomers, DNAJB1 and DNAJA2 recognize aggregation-prone conformers and even mature fibers. In addition, we find that both Hsp40s bind tau seeds and fibers via their C-terminal domain II (CTDII), with DNAJA2 being further capable of recognizing tau monomers by a second, distinct site in CTDI. These results lay out the mechanisms by which the diverse members of the Hsp40 family counteract the formation and propagation of toxic tau aggregates, and highlight the fact that chaperones from different families/classes play distinct, yet complementary roles in preventing pathological protein aggregation.
It was shown previously that the matrix (M), phosphoprotein (P), and fusion (F) proteins of respiratory syncytial virus (RSV) are sufficient to produce virus-like particles (VLPs) that resemble the RSV infection-induced virions. However, the exact mechanism and interactions among the three proteins are not known. This work examines the interaction between P and M during RSV assembly and budding. We show that M interacts with P in the absence of other viral proteins in cells by using a split Nano luciferase assay. By using recombinant proteins, we demonstrate a direct interaction between M and P. By using nuclear magnetic resonance (NMR), we identify three novel M interaction sites on P, namely, site I in the αN2 region, site II in the 115 to 125 region, and the oligomerization domain (OD). We show that the OD, and likely the tetrameric structural organization of P, is required for virus-like filament formation and VLP release. Although sites I and II are not required for VLP formation, they appear to modulate P levels in RSV VLPs.
The ubiquitous heat shock protein 70 (HSP70) family consists of ATP-dependent molecular chaperones, which perform numerous cellular functions that affect almost all aspects of the protein life cycle from synthesis to degradation1–3. Achieving this broad spectrum of functions requires precise regulation of HSP70 activity. Proteins of the HSP40 family, also known as J-domain proteins (JDPs), have a key role in this process by preselecting substrates for transfer to their HSP70 partners and by stimulating the ATP hydrolysis of HSP70, leading to stable substrate binding3,4. In humans, JDPs constitute a large and diverse family with more than 40 different members2, which vary in their substrate selectivity and in the nature and number of their client-binding domains5. Here we show that JDPs can also differ fundamentally in their interactions with HSP70 chaperones. Using nuclear magnetic resonance spectroscopy6,7 we find that the major class B JDPs are regulated by an autoinhibitory mechanism that is not present in other classes. Although in all JDPs the interaction of the characteristic J-domain is responsible for the activation of HSP70, in DNAJB1 the HSP70-binding sites in this domain are intrinsically blocked by an adjacent glycine-phenylalanine rich region—an inhibition that can be released upon the interaction of a second site on DNAJB1 with the HSP70 C-terminal tail. This regulation, which controls substrate targeting to HSP70, is essential for the disaggregation of amyloid fibres by HSP70–DNAJB1, illustrating why no other class of JDPs can substitute for class B in this function. Moreover, this regulatory layer, which governs the functional specificities of JDP co-chaperones and their interactions with HSP70s, could be key to the wide range of cellular functions of HSP70.
Hyperpolarized water can be a valuable aid in protein NMR, leading to amide group H-1 polarizations that are orders of magnitude larger than their thermal counterparts. Suitable procedures can exploit this to deliver 2D H-1-N-15 correlations with good resolution and enhanced sensitivity. These enhancements depend on the exchange rates between the amides and the water, thereby yielding diagnostic information about solvent accessibility. This study applied this "HyperW" method to four proteins exhibiting a gamut of exchange behaviors: PhoA((330-471)), an unfolded 122-residue fragment; barstar, a fully folded ribonuclease inhibitor; R17, a 13.3 kDa system possessing folded and unfolded forms under slow interconversion; and drkN SH3, a protein domain whose folded and unfolded forms interchange rapidly and with temperature-dependent population ratios. For PhoA((330-471)) HyperW sensitivity enhancements were >= 300X, as expected for an unfolded protein sequence. Though fully folded, barstar also exhibited substantial enhancements; these, however, were not uniform and, according to CLEANEX experiments, reflected the solvent-exposed residues. R17 showed the expected superposition of >= 100-fold enhancements for its unfolded form, coexisting with more modest enhancements for their folded counterparts. Unexpected, however, was the behavior of drkN SH3, for which HyperW enhanced the unfolded but, surprisingly, enhanced even more certain folded protein sites. These preferential enhancements were repeatedly and reproducibly observed. A number of explanations-including three-site exchange magnetization transfers between water and the unfolded and folded states; cross-correlated relaxation processes from hyperpolarized "structural" waters and labile side-chain protons; and the possibility that faster solvent exchange rates characterize certain folded sites over their unfolded counterparts-are considered to account for them.
Hsp70s are ubiquitous molecular chaperones that act in a myriad of cellular functions, affecting virtually all aspects in the life of proteins from synthesis to degradation. Hsp70 proteins act in the cell in cooperation with a large set of dedicated co-chaperones consisting of J-domain proteins and nucleotide exchange factors that regulate the Hsp70 chaperone cycle. Recent studies have made significant progress towards obtaining a better understanding of the mechanisms through which Hsp70s and their co-chaperones operate, providing insights into structural, kinetic, and functional features of the various members of this network. In this chapter we describe the emerging working principles of the Hsp70 machine and its co-chaperones, and highlight how mechanistic aspects of this network are tied to distinct protein folding functions.
The 70-kDa heat shock proteins (Hsp70s) are ubiquitous molecular chaperones that act in a large variety of cellular protein folding and remodelling processes. They function virtually at all stages of the life of proteins from synthesis to degradation and are thus crucial for maintaining protein homeostasis, with direct implications for human health. A large set of co-chaperones comprising J-domain proteins and nucleotide exchange factors regulate the ATPase cycle of Hsp70s, which is allosterically coupled to substrate binding and release. Moreover, Hsp70s cooperate with other cellular chaperone systems including Hsp90, Hsp60 chaperonins, small heat shock proteins and Hsp100 AAA+ disaggregases, together constituting a dynamic and functionally versatile network for protein folding, unfolding, regulation, targeting, aggregation and disaggregation, as well as degradation. In this Review we describe recent advances that have increased our understanding of the molecular mechanisms and working principles of the Hsp70 network. This knowledge showcases how the Hsp70 chaperone system controls diverse cellular functions, and offers new opportunities for the development of chemical compounds that modulate disease-related Hsp70 activities.
Large protein machines are tightly regulated through allosteric communication channels. Here we demonstrate the involvement of ultrafast conformational dynamics in allosteric regulation of CIpB, a hexameric AAA+ machine that rescues aggregated proteins. Each subunit of CIpB contains a unique coiled-coil structure, the middle domain (M domain), proposed as a control element that binds the co-chaperone DnaK. Using single-molecule FRET spectroscopy, we probe the M domain during the chaperone cycle and find it to jump on the microsecond time scale between two states, whose structures are determined. The M-domain jumps are much faster than the overall activity of CIpB, making it an effectively continuous, tunable switch. Indeed, a series of allosteric interactions are found to modulate the dynamics, including binding of nucleotides, DnaK and protein substrates. This mode of dynamic control enables fast cellular adaptation and may be a general mechanism for the regulation of cellular machineries.
Hsp70 chaperone systems are very versatile machines present in nearly all living organisms and in nearly all intracellular compartments. They function in many fundamental processes through their facilitation of protein (re)folding, trafficking, remodeling, disaggregation, and degradation. Hsp70 machines are regulated by co-chaperones. J-domain containing proteins (JDPs) are the largest family of Hsp70 co-chaperones and play a determining role functionally specifying and directing Hsp70 functions. Many features of JDPs are not understood; however, a number of JDP experts gathered at a recent CSSI-sponsored workshop in Gdansk (Poland) to discuss various aspects of J-domain protein function, evolution, and structure. In this report, we present the main findings and the consensus reached to help direct future developments in the field of Hsp70 research.
Cross-relaxation and isotropic mixing phenomena leading to the Nuclear Overhauser Effect (NOE) and to the TOCSY experiment, lie at the center of structural determinations by NMR. 2D TOCSY and NOESY exploit these polarization transfer effects to determine inter-site connectivities and molecular geometries under physiologically-relevant conditions. Among these sequences’ drawback, particularly for the case of NOEs, are a lack of sensitivity arising from small structurally-relevant cross peaks. The present study explores the application of multiple Zeno-like projective measurements, to enhance the cross-peaks between spectrally distinct groups in proteins –in particular between amide and aliphatic protons. The enhancement is based on repeating the projection done by Ramsey or TOCSY blocks multiple times, in what we refer to as Looped, PROjected Spectroscopy (L-PROSY). This leads to a reset of the amide/aliphatic transfer processes; the initial slopes of the NOE- or J-transfer effects thus define the cross-peak growth, and a faster cross-peak buildup is achieved upon looping these transfers over the allotted time T1. These projections also help to better preserve the magnetization originating in the amides, resulting in an overall improvement in sensitivity. L-PROSY's usefulness is demonstrated by incorporating it into two widely used protein NMR experiments: 2D 15N-1H HMQC-NOESY and 15N-filtered 2D NOESY. Different parameters dictating the overall SNR improvement, particularly the protein correlation times and the amide-water chemical exchange rates, were examined, and L-PROSY's enhancements resulted for all tested proteins. The largest cross-peak enhancements were observed for unstructured proteins, where chemical exchanges with the solvent of the kind that tend to average out NOE cross-peaks in conventional NMR, boost L-PROSY's cross-peaks by replenishing the amide's magnetizations within each loop. Enhanced cross-peaks were also found in extensions involving TOCSY-based experiments when applied to proteins with unfolded segments.
Molecular recognition is integral to biological function and frequently involves preferred binding of a molecule to one of several exchanging ligand conformations in solution. In such a process the bound structure can be selected from the ensemble of interconverting ligands a priori (conformational selection, CS) or may form once the ligand is bound (induced fit, IF). Here we focus on the ubiquitous and conserved Hsp70 chaperone which oversees the integrity of the cellular proteome through its ATP-dependent interaction with client proteins. We directly quantify the flux along CS and IF pathways using solution NMR spectroscopy that exploits a methyl TROSY effect and selective isotope-labeling methodologies. Our measurements establish that both bacterial and human Hsp70 chaperones interact with clients by selecting the unfolded state from a pre-existing array of interconverting structures, suggesting a conserved mode of client recognition among Hsp70s and highlighting the importance of molecular dynamics in this recognition event.
The Hsp70 chaperone system plays a critical role in cellular homeostasis by binding to client protein molecules. We have recently shown by methyl-TROSY NMR methods that the Escherichia coli Hsp70, DnaK, can form multiple bound complexes with a small client protein, hTRF1. In an effort to characterize the interactions further we report here the results of an NMR-based titration study of hTRF1 and DnaK, where both molecular components are monitored simultaneously, leading to a binding model. A central finding is the formation of a previously undetected 3:1 hTRF1-DnaK complex, suggesting that under heat shock conditions, DnaK might be able to protect cytosolic proteins whose net concentrations would exceed that of the chaperone. Moreover, these results provide new insight into the heterogeneous ensemble of complexes formed by DnaK chaperones and further emphasize the unique role of NMR spectroscopy in obtaining information about individual events in a complex binding scheme by exploiting a large number of probes that report uniquely on distinct binding processes.
The Hsp70 chaperone system is integrated into a myriad of biochemical processes that are critical for cellular proteostasis. Although detailed pictures of Hsp70 bound with peptides have emerged, correspondingly detailed structural information on complexes with folding-competent substrates remains lacking. Here we report a methyl-TROSY based solution NMR study showing that the Escherichia coli version of Hsp70, DnaK, binds to as many as four distinct sites on a small 53-residue client protein, hTRF1. A fraction of hTRF1 chains are also bound to two DnaK molecules simultaneously, resulting in a mixture of DnaK-substrate sub-ensembles that are structurally heterogeneous. The interactions of Hsp70 with a client protein at different sites results in a fuzzy chaperone-substrate ensemble and suggests a mechanism for Hsp70 function whereby the structural heterogeneity of released substrate molecules enables them to circumvent kinetic traps in their conformational free energy landscape and fold efficiently to the native state.
Ubiquitin (Ub) signaling is a diverse group of processes controlled by covalent attachment of small protein Ub and polyUb chains to a range of cellular protein targets. The best documented Ub signaling pathway is the one that delivers polyUb proteins to the 26S proteasome for degradation. However, studies of molecular interactions involved in this process have been hampered by the transient and hydrophobic nature of these interactions and the lack of tools to study them. Here, we develop Ub-phototrap (Ub(PT)), a synthetic Ub variant containing a photoactivatable crosslinking side chain. Enzymatic polymerization into chains of defined lengths and linkage types provided a set of reagents that led to identification of Rpn1 as a third proteasome ubiquitin-associating subunit that coordinates docking of substrate shuttles, unloading of substrates, and anchoring of polyUb conjugates. Our work demonstrates the value of Ub(PT), and we expect that its future uses will help define and investigate the ubiquitin interactome.
The 70-kDa heat shock protein (Hsp70) family of chaperones bind cognate substrates to perform a variety of different processes that are integral to cellular homeostasis. Although detailed structural information is available on the chaperone, the structural features of folding competent substrates in the bound form have not been well characterized. Here we use paramagnetic relaxation enhancement (PRE) NMR spectroscopy to probe the existence of long-range interactions in one such folding competent substrate, human telomere repeat binding factor (hTRF1), which is bound to DnaK in a globally unfolded conformation. We show that DnaK binding modifies the energy landscape of the substrate by removing long-range interactions that are otherwise present in the unbound, unfolded conformation of hTRF1. Because the unfolded state of hTRF1 is only marginally populated and transiently formed, it is inaccessible to standard NMR approaches. We therefore developed a H-1-based CEST experiment that allows measurement of PREs in sparse states, reporting on transiently sampled conformations. Our results suggest that DnaK binding can significantly bias the folding pathway of client substrates such that secondary structure forms first, followed by the development of longer-range contacts between more distal parts of the protein.
Solution-based NMR spectroscopy has been an important tool for studying the structure and dynamics of relatively small proteins and protein complexes with aggregate molecular masses under approximately 50 kDa. The development of new experiments and labeling schemes, coupled with continued improvements in hardware, has significantly reduced this size limitation, enabling atomic-resolution studies of molecular machines in the 1 MDa range. In this Perspective, some of the important advances are highlighted in the context of studies of molecular chaperones involved in protein disaggregation. New insights into the structural biology of disaggregation obtained from NMR studies are described, focusing on the unique capabilities of the methodology for obtaining atomic-resolution descriptions of dynamic systems.
ClpB/Hsp100 is an ATP-dependent disaggregase that solubilizes and reactivates protein aggregates in cooperation with the DnaK/Hsp70 chaperone system. The ClpB-substrate interaction is mediated by conserved tyrosine residues located in flexible loops in nucleotide-binding domain-1 that extend into the ClpB central pore. In addition to the tyrosines, the ClpB N-terminal domain (NTD) was suggested to provide a second substrate-binding site; however, the manner in which the NTD recognizes and binds substrate proteins has remained elusive. Herein, we present an NMR spectroscopy study to structurally characterize the NTD-substrate interaction. We show that the NTD includes a substrate-binding groove that specifically recognizes exposed hydrophobic stretches in unfolded or aggregated client proteins. Using an optimized segmental labeling technique in combination with methyl-transverse relaxation optimized spectroscopy (TROSY) NMR, the interaction of client proteins with both the NTD and the pore-loop tyrosines in the 580-kDa ClpB hexamer has been characterized. Unlike contacts with the tyrosines, the NTD-substrate interaction is independent of the ClpB nucleotide state and protein conformational changes that result from ATP hydrolysis. The NTD interaction destabilizes client proteins, priming them for subsequent unfolding and translocation. Mutations in the NTD substrate-binding groove are shown to have a dramatic effect on protein translocation through the ClpB central pore, suggesting that, before their interaction with substrates, the NTDs block the translocation channel. Together, our findings provide both a detailed characterization of the NTD-substrate complex and insight into the functional regulatory role of the ClpB NTD in protein disaggregation.
The 70 kDa heat shock protein (Hsp70) chaperone system is ubiquitous, highly conserved, and involved in a myriad of diverse cellular processes. Its function relies on nucleotide-dependent interactions with client proteins, yet the structural features of folding-competent substrates in their Hsp70-bound state remain poorly understood. Here we use NMR spectroscopy to study the human telomere repeat binding factor 1 (hTRF1) in complex with Escherichia coli Hsp70 (DnaK). In the complex, hTRF1 is globally unfolded with up to 40% helical secondary structure in regions distal to the binding site. Very similar conformational ensembles are observed for hTRF1 bound to ATP-, ADP- and nucleotide-free DnaK. The patterns in substrate helicity mirror those found in the unfolded state in the absence of denaturants except near the site of chaperone binding, demonstrating that DnaK-bound hTRF1 retains its intrinsic structural preferences. To our knowledge, our study presents the first atomic resolution structural characterization of a client protein bound to each of the three nucleotide states of DnaK and establishes that the large structural changes in DnaK and the associated energy that accompanies ATP binding and hydrolysis do not affect the overall conformation of the bound substrate protein.
Large macromolecular assemblies, so-called molecular machines, are critical to ensuring proper cellular function. Understanding how proper function is achieved at the atomic level is crucial to advancing multiple avenues of biomedical research. Biophysical studies often include X-ray diffraction and cryo-electron microscopy, providing detailed structural descriptions of these machines. However, their inherent flexibility has complicated an understanding of the relation between structure and function. Solution NMR spectroscopy is well suited to the study of such dynamic complexes, and continued developments have increased size boundaries; insights into function have been obtained for complexes with masses as large as 1 MDa. We highlight methyl-TROSY (transverse relaxation optimized spectroscopy) NMR, which enables the study of such large systems, and include examples of applications to several cellular machines. We show how this emerging technique contributes to an understanding of cellular function and the role of molecular plasticity in regulating an array of biochemical activities.
HSP-100 protein machines, such as ClpB, play an essential role in reactivating protein aggregates that can otherwise be lethal to cells. Although the players involved are known, including the DnaK/DnaJ/GrpE chaperone system in bacteria, details of the molecular interactions are not well understood. Using methyl-transverse relaxation-optimized nuclear magnetic resonance spectroscopy, we present an atomic-resolution model for the ClpB-DnaK complex, which we verified by mutagenesis and functional assays. ClpB and GrpE compete for binding to the DnaK nucleotide binding domain, with GrpE binding inhibiting disaggregation. DnaK, in turn, plays a dual role in both disaggregation and subsequent refolding of polypeptide chains as they emerge from the aggregate. On the basis of a combined structural-biochemical analysis, we propose a model for the mechanism of protein aggregate reactivation by ClpB.
Substrates tagged with (poly)ubiquitin for degradation can be targeted directly to the 26 S proteasome where they are proteolyzed. Independently, ubiquitin conjugates may also be delivered by bivalent shuttles. The majority of shuttles attach to the proteasome through a ubiquitin-like domain (UBL) while anchoring cargo at a C-terminal polyubiquitin-binding domain(s). We found that two shuttles of this class, Rad23 and Dsk2, dock at two different receptor sites embedded within a single subunit of the 19 S proteasome regulatory particle, Rpn1. Their association/dissociation constants and affinities for Rpn1 are similar. In contrast, another UBL-containing protein, the deubiquitinase Ubp6, is also anchored by Rpn1, yet it dissociates slower, thus behaving as an occasional proteasome subunit that is distinct from the transiently associated shuttles. Two neighboring subunits, Rpn10 and Rpn13, show a marked preference for polyubiquitin over UBLs. Rpn10 attaches to the central solenoid portion of Rpn1, although this association is stabilized by the presence of a third subunit, Rpn2. Rpn13 binds directly to Rpn2. These intrinsic polyubiquitin receptors may compete with substrate shuttles for their polyubiquitin-conjugate cargos, thereby aiding release of the emptied shuttles. By binding multiple ubiquitin-processing factors simultaneously, Rpn1 is uniquely suited to coordinate substrate recruitment, deubiquitination, and movement toward the catalytic core. The broad range of affinities for ubiquitin, ubiquitin-like, and non-ubiquitin signals by adjacent yet nonoverlapping sites all within the base represents a hub of activity that coordinates the intricate relay of substrates within the proteasome, and consequently it influences substrate residency time and commitment to degradation.
Prion diseases are associated with the conversion of cellular prion protein (PrP(C)) to toxic beta-sheet isoforms (PrP(Sc)), which are reported to inhibit the ubiquitin-proteasome system (UPS). Accordingly, UPS substrates accumulate in prion-infected mouse brains, suggesting impairment of the 26S proteasome. A direct interaction between its 20S core particle and PrP isoforms was demonstrated by immuno-precipitation. beta-PrP aggregates associated with the 20S particle, but did not impede binding of the PA26 complex, suggesting that the aggregates do not bind to its ends. Aggregated beta-PrP reduced the 20S proteasome's basal peptidase activity, and the enhanced activity induced by C-terminal peptides from the 19S ATPases or by the 19S regulator itself, including when stimulated by polyubiquitin conjugates. However, the 20S proteasome was not inhibited when the gate in the alpha-ring was open due to a truncation mutation or by association with PA26/PA28. These PrP aggregates inhibit by stabilising the closed conformation of the substrate entry channel. A similar inhibition of substrate entry into the proteasome may occur in other neurodegenerative diseases where misfolded beta-sheet-rich proteins accumulate. The EMBO Journal (2011) 30, 3065-3077. doi:10.1038/emboj.2011.224; Published online 8 July 2011
Methyl groups are powerful reporters of structure, motion, and function in NMR studies of supramolecular protein assemblies. Their utility can be hindered, however, by spectral overlap, difficulties with assignment or lack of probes in biologically important regions of the molecule studied. Here we show that 13CH3-S- labeling of Cys side chains using 13C-methyl-methanethiosulfonate (13C-MMTS) (IUPAC name: methylsulfonylsulfanylmethane) provides a convenient probe of molecular structure and dynamics. The methodology is demonstrated with an application focusing on the gating residues of the Thermoplasma acidophilum proteasome, where it is shown that the 13CH3-S- label reports faithfully on the conformational heterogeneity and dynamics in this region of the complex. A second and related application involves labeling with 13C-MMTS at the N-termini of the subunits comprising the E. coli ClpP protease that reveals multiple conformations of gating residues in this complex as well. These N-terminal residues adopt a single conformation upon gate opening.
As a signal for substrate targeting, polyubiquitin meets various layers of receptors upstream to the 26S proteasome. We obtained structural information on two receptors, Rpn10 and Dsk2, alone and in complex with (poly)ubiquitin or with each other. A hierarchy of affinities emerges with Dsk2 binding monoubiquitin tighter than Rpn10 does, whereas Rpn10 prefers the ubiquitin-like domain of Dsk2 to monoubiquitin, with increasing affinities for longer polyubiquitin chains. We demonstrated the formation of ternary complexes of both receptors simultaneously with (poly)ubiquitin and found that, depending on the ubiquitin chain length, the orientation of the resulting complex is entirely different, providing for alternate signals. Dynamic rearrangement provides a chain-length sensor, possibly explaining how accessibility of Dsk2 to the proteasome is limited unless it carries a properly tagged cargo. We propose a mechanism for a malleable ubiquitin signal that depends both on chain length and combination of receptors to produce tetraubiquitin as an efficient signal threshold.
Rpn1 (109 kDa) and Rpn2 (1.04 kDa) are components of the 19S regulatory complex of the proteasome. The central portions of both proteins are predicted to have toroidal alpha-solenoid folds composed of 9-11 proteasome/cyclosome repeats, each similar to 40 residues long and containing two alpha-helices and turns [A. V. Kajava, J. Biol. Chem. 277, 49791-49798, 2002]. To evaluate this prediction, we examined the full-length yeast proteins and truncated versions thereof consisting only of the repeat-containing regions by gel filtration, CD spectroscopy, and negative-staining electron microscopy (EM). All four proteins are monomeric in solution and highly alpha-helical, particularly the truncated ones. The EM data were analyzed by image classification and averaging techniques. The preponderant projections, in each case, show near-annular molecules 6-7 nm in diameter. Comparison of the full-length with the truncated proteins showed molecules similar in size and shape, indicating that their terminal regions are flexible and thus smeared to invisibility in the averaged images. We tested the toroidal model further by calculating resolution-limited projections and comparing them with the EM images. The results support the alpha-solenoid model, except that they indicate that the repeats are organized not as symmetrical circular toroids but in less regular horseshoe-like structures. Published by Elsevier Ltd.
Assembly of the 34-subunit, 2.5 MDa 26S proteasome is a carefully choreographed intricate process. It starts with formation of a seven-membered alpha-ring that serves as a template for assembly of the complementary beta-ring-forming 'half-proteasomes'. Dimerization results in a latent 20S core particle that can serve further as a platform for 19S regulatory particle attachment and formation of the biologically active 26S proteasome for ubiquitin-dependent proteolysis. Both general and dedicated proteasome assembly chaperones regulate the efficiency and outcome of critical steps in proteasome biogenesis, and in complex association.
The 26S proteasome is a multisubunit enzyme composed of a cylindrical catalytic core (20S) and a regulatory particle (19S) that together perform the essential degradation of cellular proteins tagged by ubiquitin. To date, however, substrate trajectory within the complex remains elusive. Here we describe a previously unknown functional unit within the 19S, comprising two subunits, Rpn1 and Rpn2. These toroids physically link the site of substrate recruitment with the site of proteolysis. Rpn2 interfaces with the 20S, whereas Rpn1 sits atop Rpn2, serving as a docking site for a substrate-recruitment factor. The 19S ATPases encircle the Rpn1-Rpn2 stack, covering the remainder of the 20S surface. Both Rpn1-Rpn2 and the ATPases are required for substrate translocation and gating of the proteolytic channel. Similar pairing of units is found in unfoldases and nuclear transporters, exposing common features of these protein nanomachines.
Assembly of the 34- subunit, 2.5 megadalton 26S proteasome starts with formation of the seven-membered alpha-ring. A set of newly identified proteasome chaperones serves as a clamp to seal alpha-rings with the correct composition. By regulating the efficiency and outcome of this crucial step in proteasome biogenesis, these dedicated proteasome chaperones apparently partake in the stress response and in adaptation to intracellular proteolysis needs.