Structure and molecular dynamics simulation of archaeal prefoldin: the molecular mechanism for binding and recognition of nonnative substrate proteins

Prefoldin (PFD) is a heterohexameric molecular chaperone complex in the eukaryotic cytosol and archaea with a jellyfish-like structure containing six long coiled-coil tentacles. PFDs capture protein folding intermediates or unfolded polypeptides and transfer them to group II chaperonins for facilitated folding. Although detailed studies on the mechanisms for interaction with unfolded proteins or cooperation with chaperonins of archaeal PFD have been performed, it is still unclear how PFD captures the unfolded protein. In this study, we determined the X-ray structure of Pyrococcus horikoshii OT3 PFD (PhPFD) at 3.0 Å resolution and examined the molecular mechanism for binding and recognition of nonnative substrate proteins by molecular dynamics (MD) simulation and mutation analyses. PhPFD has a jellyfish-like structure with six long coiled-coil tentacles and a large central cavity. Each subunit has a hydrophobic groove at the distal region where an unfolded substrate protein is bound. During MD simulation at 330 K, each coiled coil was highly flexible, enabling it to widen its central cavity and capture various nonnative proteins. Docking MD simulation of PhPFD with unfolded insulin showed that the β subunit is essentially involved in substrate binding and that the α subunit modulates the shape and width of the central cavity. Analyses of mutant PhPFDs with amino acid replacement of the hydrophobic residues of the β subunit in the hydrophobic groove have shown that βIle107 has a critical role in forming the hydrophobic groove.     

Structural and molecular characterization of the prefoldin beta subunit from Thermococcus strain KS-1

Prefoldin (PFD) is a heterohexameric molecular chaperone that is found in eukaryotic cytosol and archaea. PFD is composed of α and β subunits and forms a “jellyfish-like” structure. PFD binds and stabilizes nascent polypeptide chains and transfers them to group II chaperonins for completion of their folding. Recently, the whole genome of Thermococcus kodakaraensis KOD1 was reported and shown to contain the genes of two α and two β subunits of PFD. The genome of Thermococcus strain KS-1 also possesses two sets of α (α1 and α2) and β subunits (β1 and β2) of PFD (TsPFD). However, the functions and roles of each of these PFD subunits have not been investigated in detail. Here, we report the crystal structure of the TsPFD β1 subunit at 1.9 Å resolution and its functional analysis. TsPFD β1 subunits form a tetramer with four coiled-coil tentacles resembling the jellyfish-like structure of heterohexameric PFD. The β hairpin linkers of β1 subunits assemble to form a β barrel “body” around a central fourfold axis. Size-exclusion chromatography and multi-angle light-scattering analyses show that the β1 subunits form a tetramer at pH 8.0 and a dimer of tetramers at pH 6.8. The tetrameric β1 subunits can protect against aggregation of relatively small proteins, insulin or lysozyme. The structural and biochemical analyses imply that PFD β1 subunits act as molecular chaperones in living cells of some archaea.     

Selective contribution of eukaryotic prefoldin subunits to actin and tubulin binding

Eukaryotic prefoldin (PFD) is a heterohexameric chaperone with a jellyfish-like structure whose function is to deliver nonnative target proteins, principally actins and tubulins, to the eukaryotic cytosolic chaperonin for facilitated folding. Here we demonstrate that functional PFD can spontaneously assemble from its six constituent individual subunits (PFD1-PFD6), each expressed as a recombinant protein. Using engineered forms of PFD assembled in vitro, we show that the tips of the PFD tentacles are required to form binary complexes with authentic target proteins. We show that PFD uses the distal ends of different but overlapping sets of subunits to form stable binary complexes with different target proteins, namely actin and alpha- and beta-tubulin. We also present data that suggest a model for the order of these six subunits within the hexamer. Our data are consistent with the hypothesis that PFD, like the eukaryotic cytosolic chaperonin, has co-evolved specifically to facilitate the folding of its target proteins.     

Structure of eukaryotic prefoldin and of its complexes with unfolded actin and the cytosolic chaperonin CCT

The biogenesis of the cytoskeletal proteins actin and tubulin involves interaction of nascent chains of each of the two proteins with the oligomeric protein prefoldin (PFD) and their subsequent transfer to the cytosolic chaperonin CCT (chaperonin containing TCP-1). Here we show by electron microscopy that eukaryotic PFD, which has a similar structure to its archaeal counterpart, interacts with unfolded actin along the tips of its projecting arms. In its PFD-bound state, actin seems to acquire a conformation similar to that adopted when it is bound to CCT. Three-dimensional reconstruction of the CCT:PFD complex based on cryoelectron microscopy reveals that PFD binds to each of the CCT rings in a unique conformation through two specific CCT subunits that are placed in a 1,4 arrangement. This defines the phasing of the CCT rings and suggests a handoff mechanism for PFD.     

Prefoldin subunits are protected from ubiquitin-proteasome system-mediated degradation by forming complex with other constituent subunits

The molecular chaperone prefoldin (PFD) is a complex comprised of six different subunits, PFD1-PFD6, and delivers newly synthesized unfolded proteins to cytosolic chaperonin TRiC/CCT to facilitate the folding of proteins. PFD subunits also have functions different from the function of the PFD complex. We previously identified MM-1α/PFD5 as a novel c-Myc-binding protein and found that MM-1α suppresses transformation activity of c-Myc. However, it remains unclear how cells regulate protein levels of individual subunits and what mechanisms alter the ratio of their activities between subunits and their complex. In this study, we found that knockdown of one subunit decreased protein levels of other subunits and that transfection of five subunits other than MM-1α into cells increased the level of endogenous MM-1α. We also found that treatment of cells with MG132, a proteasome inhibitor, increased the level of transfected/overexpressed MM-1α but not that of endogenous MM-1α, indicating that overexpressed MM-1α, but not endogenous MM-1α, was degraded by the ubiquitin proteasome system (UPS). Experiments using other PFD subunits showed that the UPS degraded a monomer of PFD subunits, though extents of degradation varied among subunits. Furthermore, the level of one subunit was increased after co-transfection with the respective subunit, indicating that there are specific combinations between subunits to be stabilized. These results suggest mutual regulation of protein levels among PFD subunits and show how individual subunits form the PFD complex without degradation.     

Hyperthermophilic archaeal prefoldin shows refolding activity at low temperature

Prefoldin is a molecular chaperone that captures a protein-folding intermediate and transfers it to a group II chaperonin for correct folding. Previous studies of archaeal prefoldins have shown that prefoldin only possesses holdase activity and is unable to fold unfolded proteins by itself. In this study, we have demonstrated for the first time that a prefoldin from hyperthermophilic archaeon, Pyrococcus horikoshii OT3 (PhPFD), exhibits refolding activity for denatured lysozyme at temperatures relatively lower than physiologically active temperatures. The interaction between PhPFD and denatured lysozyme was investigated by use of a surface plasmon resonance sensor at various temperatures. Although PhPFD showed strong affinity for denatured lysozyme at high temperature, it exhibited relatively weak interactions at lower temperature. The protein-folding seems to occur through binding and release from PhPFD by virtue of the weak affinity. Our results also imply that prefoldin might be able to contribute to the folding of some cellular proteins whose affinity with prefoldin is weak.     

Eukaryotic chaperonin CCT stabilizes actin and tubulin folding intermediates in open quasi-native conformations

Three-dimensional reconstruction from cryoelectron micrographs of the eukaryotic cytosolic chaperonin CCT complexed to tubulin shows that CCT interacts with tubulin (both the alpha and beta isoforms) using five specific CCT subunits. The CCT-tubulin interaction has a different geometry to the CCT-actin interaction, and a mixture of shared and unique CCT subunits is used in binding the two substrates. Docking of the atomic structures of both actin and tubulin to their CCT-bound conformation suggests a common mode of chaperonin-substrate interaction. CCT stabilizes quasi-native structures in both proteins that are open through their domain-connecting hinge regions, suggesting a novel mechanism and function of CCT in assisted protein folding.     

ATP hydrolysis by the proteasome regulatory complex PAN serves multiple functions in protein degradation

To clarify the role of ATP in proteolysis, we studied archaeal 20S proteasomes and the PAN (proteasome-activating nucleotidase) regulatory complex, a homolog of the eukaryotic 19S ATPases. PAN's ATPase activity was stimulated similarly by globular (GFPssrA) and unfolded (casein) substrates, and by the ssrA recognition peptide. Denaturation of GFPssrA did not accelerate its degradation or eliminate the requirement for PAN and ATP. During degradation of one molecule of globular or unfolded substrates, 300-400 ATP molecules were hydrolyzed. An N-terminal deletion in the 20S alpha subunits caused opening of the substrate-entry channel and rapid degradation of unfolded proteins without PAN; however, degradation of globular GFPssrA still required PAN's ATPase activity, even after PAN-catalyzed unfolding. Thus, substrate binding activates ATP hydrolysis, which promotes three processes: substrate unfolding, gate opening in the 20S, and protein translocation.     

N- and C-Terminal motifs in human alphaB crystallin play an important role in the recognition, selection, and solubilization of substrates

The functions of the interactive sequences in human alphaB crystallin that are involved in chaperone activity and complex assembly of small heat shock proteins need to be characterized to understand the mechanisms of action on unfolding and misfolding proteins. Protein pin arrays identified the hydrophobic N-terminal sequence (41STSLSPFYLRPPSFLRAP58) and the polar C-terminal sequence (155PERTIPITREE165) as interactive domains in human alphaB crystallin, which were then deleted to evaluate their importance in complex assembly and chaperone activity. Size exclusion chromatography determined that the complexes formed by the deletion mutants, Delta41-58 and Delta155-165, were larger and more polydisperse than the wild-type (wt) alphaB crystallin complex. In chaperone assays, the Delta41-58 mutant was as effective as wt alphaB crystallin in protecting partially unfolded betaL crystallin and alcohol dehydrogenase (ADH) and significantly less effective than wt alphaB crystallin in protecting unfolded citrate synthase (CS) from aggregation. Chaperone activity did not correlate with complex size but corresponded with the amount of substrate protein unfolding. The results confirmed the importance of N-terminal residues 41-58 in selective interactions with completely unfolded substrates. Poor solubility and limited or no chaperone activity for the three substrates characterized the Delta155-165 deletion mutant, which demonstrated the importance of C-terminal residues 155-165 in maintaining the solubility of unfolded substrates in a manner independent of the amount of substrate protein unfolding. The results presented in this report established that interactive domains in the N- and C-termini of human alphaB crystallin are important for the recognition, selection, and solubility of unfolding substrate proteins.     

Substrate transfer from the chaperone Hsp70 to Hsp90

Hsp90 is an essential chaperone protein in the cytosol of eukaryotic cells. It cooperates with the chaperone Hsp70 in defined complexes mediated by the adaptor protein Hop (Sti1 in yeast). These Hsp70/Hsp90 chaperone complexes play a major role in the folding and maturation of key regulatory proteins in eukaryotes. Understanding how non-native client proteins are transferred from one chaperone to the other in these complexes is of central importance. Here, we analyzed the molecular mechanism of this reaction using luciferase as a substrate protein. Our experiments define a pathway for luciferase folding in the Hsp70/Hsp90 chaperone system. They demonstrate that Hsp70 is a potent capture device for unfolded protein while Hsp90 is not very efficient in this reaction. When Hsp90 is absent, in contrast to the in vivo situation, Hsp70 together with the two effector proteins Ydj1 and Sti1 exhibits chaperone activity towards luciferase. In the presence of the complete chaperone system, Hsp90 exhibits a specific positive effect only in the presence of Ydj1. If this factor is absent, the transferred luciferase is trapped on Hsp90 in an inactive conformation. Interestingly, identical results were observed for the yeast and the human chaperone systems although the regulatory function of human Hop is completely different from that of yeast Sti1.     

Partially Folded Aggregation Intermediates of Human γD-, γC-, and γS-Crystallin Are Recognized and Bound by Human αB-Crystallin Chaperone

Human γ-crystallins are long-lived, unusually stable proteins of the eye lens exhibiting duplicated, double Greek key domains. The lens also contains high concentrations of the small heat shock chaperone α-crystallin, which suppresses aggregation of model substrates in vitro. Mature-onset cataract is believed to represent an aggregated state of partially unfolded and covalently damaged crystallins. Nonetheless, the lack of cell or tissue culture for anucleate lens fibers and the insoluble state of cataract proteins have made it difficult to identify the conformation of the human γ-crystallin substrate species recognized by human α-crystallin. The three major human lens monomeric γ-crystallins, γD, γC, and γS, all refold in vitro in the absence of chaperones, on dilution from denaturant into buffer. However, off-pathway aggregation of the partially folded intermediates competes with productive refolding. Incubation with human αB-crystallin chaperone during refolding suppressed the aggregation pathways of the three human γ-crystallin proteins. The chaperone did not dissociate or refold the aggregated chains under these conditions. The αB-crystallin oligomers formed long-lived stable complexes with their γD-crystallin substrates. Using α-crystallin chaperone variants lacking tryptophans, we obtained fluorescence spectra of the chaperone-substrate complex. Binding of substrate γ-crystallins with two or three of the four buried tryptophans replaced by phenylalanines showed that the bound substrate remained in a partially folded state with neither domain native-like. These in vitro results provide support for protein unfolding/protein aggregation models for cataract, with α-crystallin suppressing aggregation of damaged or unfolded proteins through early adulthood but becoming saturated with advancing age.     

Functional TIM10 chaperone assembly is redox-regulated in vivo

The TIM10 chaperone facilitates the insertion of hydrophobic proteins at the mitochondrial inner membrane. Here we report the novel molecular mechanism of TIM10 assembly. This process crucially depends on oxidative folding in mitochondria and involves: (i) import of the subunits in a Cys-reduced and unfolded state; (ii) folding to an assembly-competent structure maintained by intramolecular disulfide bonding of their four conserved cysteines; and (iii) assembly of the oxidized zinc-devoid subunits to the functional complex. We show that intramolecular disulfide bonding occurs in vivo, whereas intermolecular disulfides observed in vitro are abortive intermediates in the assembly pathway. This novel mechanism of compartment-specific redox-regulated assembly is crucial for the formation of a functional TIM10 chaperone.     

A filamentous molecular chaperone of the prefoldin family from the deep-sea hyperthermophile Methanocaldococcus jannaschii

Prefoldin is a molecular chaperone found in the domains eukarya and archaea that acts in conjunction with Group II chaperonin to correctly fold other nascent proteins. Previously, our group identified a putative single subunit of prefoldin, gamma PFD, that was up-regulated in response to heat stress in the hyperthermophilic archaeon Methanocaldococcus jannaschii. In order to characterize this protein, we subcloned and expressed it and the other two prefoldin subunits from M. jannaschii, alpha and beta PFD, into Eschericia coli and characterized the proteins. Whereas alpha and beta PFD readily assembled into the expected hexamer, gamma PFD would not assemble with either protein. Instead, gamma PFD forms long filaments of defined dimensions measuring 8.5 nm x 1.7-3.5 nm and lengths exceeding 1 microm. Filamentous gamma PFD acts as a molecular chaperone through in vitro assays, in a manner comparable to PFD. A possible molecular model for filament assembly is discussed.     

The Large Hsp70 Grp170 Binds to Unfolded Protein Substrates in Vivo with a Regulation Distinct from Conventional Hsp70s

The Hsp70 superfamily is a ubiquitous chaperone class that includes conventional and large Hsp70s. BiP is the only conventional Hsp70 in the endoplasmic reticulum (ER) whose functions include: assisting protein folding, targeting misfolded proteins for degradation, and regulating the transducers of the unfolded protein response. The ER also possesses a single large Hsp70, the glucose-regulated protein of 170 kDa (Grp170). Like BiP it is an essential protein, but its cellular functions are not well understood. Here we show that Grp170 can bind directly to a variety of incompletely folded protein substrates in the ER, and as expected for a bona fide chaperone, it does not interact with folded secretory proteins. Our data demonstrate that Grp170 and BiP associate with similar molecular forms of two substrate proteins, but while BiP is released from unfolded substrates in the presence of ATP, Grp170 remains bound. In comparison to conventional Hsp70s, the large Hsp70s possess two unique structural features: an extended C-terminal α-helical domain and an unstructured loop in the putative substrate binding domain with an unknown function. We find that in the absence of the α-helical domain the interaction of Grp170 with substrates is reduced. In striking contrast, deletion of the unstructured loop results in increased binding to substrates, suggesting the presence of unique intramolecular mechanisms of control for the chaperone functions of large Hsp70s.     

Roles of the N-domains of the ClpA unfoldase in binding substrate proteins and in stable complex formation with the ClpP protease

The hexameric cylindrical Hsp100 chaperone ClpA mediates ATP-dependent unfolding and translocation of recognized substrate proteins into the coaxially associated serine protease ClpP. Each subunit of ClpA is composed of an N-terminal domain of approximately 150 amino acids at the top of the cylinder followed by two AAA+ domains. In earlier studies, deletion of the N-domain was shown to have no effect on the rate of unfolding of substrate proteins bearing a C-terminal ssrA tag, but it did reduce the rate of degradation of these proteins (Lo, J. H., Baker, T. A., and Sauer, R. T. (2001) Protein Sci. 10, 551-559; Singh, S. K., Rozycki, J., Ortega, J., Ishikawa, T., Lo, J., Steven, A. C., and Maurizi, M. R. (2001) J. Biol. Chem. 276, 29420-29429). Here we demonstrate, using both fluorescence resonance energy transfer to measure the arrival of substrate at ClpP and competition between wild-type and an inactive mutant form of ClpP, that this effect on degradation is caused by diminished stability of the ClpA-ClpP complex during translocation and proteolysis, effectively disrupting the targeting of unfolded substrates to the protease. We have also examined two larger ssrA-tagged substrates, CFP-GFP-ssrA and luciferase-ssrA, and observed different behaviors. CFP-GFP-ssrA is not efficiently unfolded by the truncated chaperone whereas luciferase-ssrA is, suggesting that the former requires interaction with the N-domains, likely via the body of the protein, to stabilize its binding. Thus, the N-domains play a key allosteric role in complex formation with ClpP and may also have a critical role in recognizing certain tag elements and binding some substrate proteins.     

Crystal structure of Skp, a prefoldin-like chaperone that protects soluble and membrane proteins from aggregation

The Seventeen Kilodalton Protein (Skp) is a trimeric periplasmic chaperone that assists outer membrane proteins in their folding and insertion into membranes. Here we report the crystal structure of Skp from E. coli. The structure of the Skp trimer resembles a jellyfish with alpha-helical tentacles protruding from a beta barrel body defining a central cavity. The architecture of Skp is unexpectedly similar to that of Prefoldin/GimC, a cytosolic chaperone present in eukaria and archea, that binds unfolded substrates in its central cavity. The ability of Skp to prevent the aggregation of model substrates in vitro is independent of ATP. Skp can interact directly with membrane lipids and lipopolysaccharide (LPS). These interactions are needed for efficient Skp-assisted folding of membrane proteins. We have identified a putative LPS binding site on the outer surface of Skp and propose a model for unfolded substrate binding.     

MtGimC, a novel archaeal chaperone related to the eukaryotic chaperonin cofactor GimC/prefoldin

Group II chaperonins in the eukaryotic and archaeal cytosol assist in protein folding independently of the GroES-like cofactors of eubacterial group I chaperonins. Recently, the eukaryotic chaperonin was shown to cooperate with the hetero-oligomeric protein complex GimC (prefoldin) in folding actin and tubulins. Here we report the characterization of the first archaeal homologue of GimC, from Methanobacterium thermoautotrophicum. MtGimC is a hexamer of 87 kDa, consisting of two alpha and four beta subunits of high alpha-helical content that are predicted to contain extended coiled coils and represent two evolutionarily conserved classes of Gim subunits. Reconstitution experiments with MtGimC suggest that two subunits of the alpha class (archaeal Gimalpha and eukaryotic Gim2 and 5) form a dimer onto which four subunits of the beta class (archaeal Gimbeta and eukaryotic Gim1, 3, 4 and 6) assemble. MtGimalpha and beta can form hetero-complexes with yeast Gim subunits and MtGimbeta partially complements yeast strains lacking Gim1 and 4. MtGimC is a molecular chaperone capable of stabilizing a range of non-native proteins and releasing them for subsequent chaperonin-assisted folding. In light of the absence of Hsp70 chaperones in many archaea, GimC may fulfil an ATP-independent, Hsp70-like function in archaeal de novo protein folding.     

Dancing with the Diva: Hsp90–Client Interactions

The molecular chaperone Hsp90 is involved in the folding, maturation, and degradation of a large number structurally and sequentially unrelated clients, often connected to serious diseases. Elucidating the principles of how Hsp90 recognizes this large variety of substrates is essential for comprehending the mechanism of this chaperone machinery, as well as it is a prerequisite for the design of client specific drugs targeting Hsp90. Here, we discuss the recent progress in understanding the substrate recognition principles of Hsp90 and its implications for the role of Hsp90 in the lifecycle of proteins. Hsp90 acts downstream of the chaperone Hsp70, which exposes its substrate to a short and highly hydrophobic cleft. The subsequently acting Hsp90 has an extended client-binding interface that enables a large number of low-affinity contacts. Structural studies show interaction modes of Hsp90 with the intrinsically disordered Alzheimer's disease-causing protein Tau, the kinase Cdk4 in a partially unfolded state and the folded ligand-binding domain of a steroid receptor. Comparing the features shared by these different proteins provides a picture of the substrate-binding principles of Hsp90.     

Binding and degradation of heterodimeric substrates by ClpAP and ClpXP

ClpA and ClpX function both as molecular chaperones and as the regulatory components of ClpAP and ClpXP proteases, respectively. ClpA and ClpX bind substrate proteins through specific recognition signals, catalyze ATP-dependent protein unfolding of the substrate, and when in complexes with ClpP translocate the unfolded polypeptide into the cavity of the ClpP peptidase for degradation. To examine the mechanism of interaction of ClpAP with dimeric substrates, single round binding and degradation experiments were performed, revealing that ClpAP degraded both subunits of a RepA homodimer in one cycle of binding. Furthermore, ClpAP was able to degrade both protomers of a RepA heterodimer in which only one subunit contained the ClpA recognition signal. In contrast, ClpXP degraded both subunits of a dimeric substrate only when both protomers contained a recognition signal. These data suggest that ClpAP and ClpXP may recognize and bind substrates in significantly different ways.