Purification and characterization of prokaryotic and eukaryotic Hsp90.
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Bacterial Proteins↗
Biomedical subjects
J Buchner
Publications and source records attributed to J Buchner.
Purification of Hsp90 partner proteins Hop/p60, p23, and FKBP52.
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Animals↗
How chaperones fold proteins.
Chaperones are a functionally related group of proteins assisting protein folding in the cell under physiological and stress conditions. They share the ability to recognize and bind nonnative proteins thus preventing unspecific aggregation. The underlying functional principles of the different chaperone classes are beginning to be understood. A landmark feature of molecular chaperones is the involvement of energy-dependent reactions in the folding process. Nucleotide binding to ATP-dependent chaperones (e.g. GroEL, Hsp70, Hsp90) leads to sometimes large conformational changes in the chaperone which allow to shift between high- and low-affinity states for substrate proteins. Interestingly, the ATPase activity which is the key determinant for functional cycles is tightly regulated by a set of co-chaperones. While for ATP-dependent chaperones binding sites for nucleotide and protein are found in one protein, in the case of ATP-independent chaperones (e. g. sHsps, SecB) the energy-dependent step is performed by another chaperone (Hsp70, SecA). Therefore, the ATP-independent chaperones can be regarded as efficient 'holding' components. Cooperation of different chaperone machineries creates a synergistic network of folding helpers in the cell, which allows to maintain protein homeostasis under conditions nonpermissive for spontaneous folding.
Models, Chemical↗
ATP-binding properties of human Hsp90.
Hsp90 is one of the most abundant proteins in the cytosol of eukaryotic cells. Under physiological conditions Hsp90 has been shown to play a major role in several specific signaling pathways, including maturation of various kinases and maintenance of steroid receptors in an activable state. It is well established that the level of Hsp90 increases severalfold under stress conditions, and it has been shown that the chaperone function of Hsp90 is ATP-independent. Although yeast Hsp90 does not bind ATP, as determined by a number of methods monitoring tight binding, ATP-dependent functions of Hsp90 in the presence of co-factors and elevated temperatures are still under discussion. Here, we have reinvestigated ATP-binding properties and ATPase activity of human Hsp90 under various conditions. We show that human Hsp90 does not bind ATP tightly and does not exhibit detectable ATPase activity. However, using electron spin resonance spectroscopy, weak binding of spin-labeled ATP analogues with half-maximal binding at 400 microM ATP was detected. The functional significance of this weak interaction remains enigmatic.
Adenosine Triphosphatases↗
How GroES regulates binding of nonnative protein to GroEL.
At present, it is still enigmatic how the reaction cycle by which the Escherichia coli GroE chaperones mediate protein folding in the cell is coordinated with respect to the sequential order of binding and release of GroES, nucleotide, and nonnative protein. It is generally assumed that the asymmetric GroEL.GroES complex is the acceptor state for substrate protein. Nevertheless, this species is poorly understood in its binding characteristics for nucleotide and nonnative protein. We show here that this species has a high affinity binding site for nonnative protein. In addition to this, binding of nucleotide to one GroEL ring is strongly favored by GroES binding to the other ring. However, the slow rate of release of substrate protein from the unproductive trans-position kinetically favors the binding of a second GroES, thereby forming a symmetric GroEL14.(GroES7)2 complex and simultaneously ensuring that substrate protein is sequestered in a position underneath GroES. Our results demonstrate that the intrinsic binding characteristics of the trans-bullet complex determine the sequence of events during the reaction cycle.
Bacterial Proteins↗
Catalysis of protein folding by symmetric chaperone complexes.
The GroE chaperones of Escherichia coli assist protein folding under physiological and heat shock conditions in an ATP-dependent way. Although a number of details of assisted folding have been elucidated, the molecular mechanism of the GroE cycle remains unresolved. Here we present an experimental system that allows the direct analysis of the GroE-mediated folding cycle under stringent conditions. We demonstrate that the GroE proteins efficiently catalyze the folding of kinetically trapped folding intermediates of a mutant of maltose-binding protein (MBP Y283D) in an ATP-dependent way. GroES plays a key role in this reaction cycle, accelerating the folding of the substrate protein MBP Y283D up to 50-fold. Interestingly, catalysis of the folding reaction requires the formation of symmetrical football-shaped GroEL x GroES2 particles and the intermediate release of the nonnative protein from the chaperone complex. Our results show that, in the presence of GroES, the complex architecture of the GroEL toroids allows maintenance of two highly interregulated rings simultaneously active in protein folding.
ATP-Binding Cassette Transporters↗
Binding of non-native protein to Hsp25 during heat shock creates a reservoir of folding intermediates for reactivation.
Small heat shock proteins (sHsps) are a conserved and ubiquitous protein family. Their ability to convey thermoresistance suggests their participation in protecting the native conformation of proteins. However, the underlying functional principles of their protective properties and their role in concert with other chaperone families remain enigmatic. Here, we analysed the influence of Hsp25 on the inactivation and subsequent aggregation of a model protein, citrate synthase (CS), under heat shock conditions in vitro. We show that stable binding of several non-native CS molecules to one Hsp25 oligomer leads to an accumulation of CS unfolding intermediates, which are protected from irreversible aggregation. Furthermore, a number of different proteins which bind to Hsp25 can be isolated from heat-shocked extracts of cells. Under permissive folding conditions, CS can be released from Hsp25 and, in cooperation with Hsp70, an ATP-dependent chaperone, the native state can be restored. Taken together, our findings allow us to integrate sHsps functionally in the cellular chaperone system operating under heat shock conditions. The task of sHsps in this context is to efficiently trap a large number of unfolding proteins in a folding-competent state and thus create a reservoir of non-native proteins for an extended period of time, allowing refolding after restoration of physiological conditions in cooperation with other chaperones.
Animals↗
Chaperone function of Hsp90-associated proteins.
The Hsp90 heat shock protein of eukaryotic cells regulates the activity of proteins involved in signal transduction pathways and may direct intracellular protein folding in general. Hsp90 performs at least part of its function in a complex with a specific set of partner proteins that include members of the prolyl isomerase family. The properties of the major components of the Hsp90 complex were examined through the use of in vitro protein folding assays. Two of the components, FKBP52 and p23, functioned as mechanistically distinct molecular chaperones. These results suggest the existence of a super-chaperone complex in the cytosol of eukaryotic cells.
Amino Acid Isomerases↗
Dynamics of the GroEL-protein complex: effects of nucleotides and folding mutants.
Chaperonins are a ubiquitous class of ring-shaped oligomeric protein complexes that are of crucial importance for protein folding in vivo. Analysis of the underlying functional principles had relied mainly on model proteins the (un)folding of which is dominated by irreversible side-reactions. We used maltose-binding protein (MBP) as a substrate protein for GroEL, since the refolding of this protein is completely reversible and thus allows a detailed analysis of the molecular parameters that determine the interaction of GroEL with non-native protein. We show that MBP folding intermediates are effectively trapped by GroEL in a diffusion-controlled reaction. This complex is stabilized via unspecific hydrophobic interactions. Stabilization energies for wild-type MBP increasing linearly with ionic strength from 50 kJ/mol to 60 kJ/mol. Depending on the intrinsic folding rate and the hydrophobicity of the substrate protein, the interaction of GroEL with MBP folding intermediates leads to a dramatically decreased apparent refolding rate of MBP (wild-type) or a complete suppression of folding (MBP folding mutant Y283D). On the basis of our data, a quantitative kinetic model of the GroEL-mediated folding cycle is proposed, which allows simulation of the partial reactions of the binding and release cycles under all conditions tested. In the presence of ATP and non-hydrolysable analogues, MBP is effectively released from GroEL, since the overall dissociation constant is reduced by three orders of magnitude. Interestingly, binding of nucleotide does not change the off rate by more than a factor of 3. However the on-rate is decreased by at least two orders of magnitude. Therefore, the rebinding reaction is prevented and folding occurs in solution.
Adenine Nucleotides↗
Assessment of the ATP binding properties of Hsp90.
Hsp90, one of the most prominent proteins in eucaryotic cells under physiological and stress conditions, chaperones protein folding reactions in an ATP-independent way. Surprisingly, ATP binding and ATPase activity of Hsp90 has been reported by several groups. To clarify this important issue, we have reinvestigated the potential ATP binding properties and ATPase activity of highly purified Hsp90 using a number of different techniques. Hsp90 was compared to the well characterized ATP-binding chaperone Hsc70 and to two control proteins, immunoglobulin G and bovine serum albumin, that are known to not bind ATP. Hsp90 behaved very similarly to the non-ATP-binding proteins and very differently from the ATP-binding protein Hsc70. Like bovine serum albumin and immunoglobulin G, Hsp90 (i) did not bind to immobilized ATP, (ii) could not be specifically photocross-linked with azido-ATP, (iii) failed to exhibit significant changes in intrinsic protein fluorescence upon ATP addition, and (iv) did not bind to three fluorescent ADP analogues. In contrast, Hsc70 strongly bound ATP and ADP, specifically cross-linked with azido-ATP, and exhibited major shifts in fluorescence upon addition of ATP. Finally, reexamination of the amino acid sequence of Hsp90 failed to reveal any significant homologies to known ATP-binding motifs. Taken together, we conclude that highly purified Hsp90 does not bind ATP. Weak ATPase activities associated with Hsp90 preparations may be due to minor impurities or kinases copurifying with Hsp90.
Adenosine Diphosphate↗
Chaperone function on Crete: a meeting report.
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Animals↗
Supervising the fold: functional principles of molecular chaperones.
Molecular chaperones are a set of conserved protein families that share the remarkable ability to recognize and selectively bind nonnative proteins under physiological and stress conditions. Thus, they prevent irreversible aggregation reactions and keep proteins on the productive folding pathway. Evidence suggests that the cell has developed several functionally distinct chaperone families to support protein folding. The importance of molecular chaperones under stress conditions is highlighted by the finding that all the major heat shock protein families (Hsp104, Hsp90, Hsp70, Hsp60/GroEL, and small Hsps) suppress irreversible unfolding reactions. Under heat shock, only the increased expression of a repertoire of different chaperones seems to convey thermotolerance and guarantee survival. The molecular mechanism by which chaperones in general influence protein folding processes is still far from clear. However, significant progress has been achieved in understanding some of the partial reactions of the chaperone folding cycles and in functionally differentiating between the different chaperone families.
Bacterial Proteins↗
BiP binding sequences in antibodies.
During the process of folding and assembly of antibody molecules in the endoplasmic reticulum, immunoglobulin heavy and light chains associate transiently with BiP, a resident endoplasmic reticulum protein that is a member of the Hsp70 family of molecular chaperones. BiP is thought to recognize unfolded or unassembled polypeptides by binding extended sequences of approximately seven amino acids that include bulky hydrophobic residues not normally exposed on the surface of native proteins. We used a computer algorithm developed to predict BiP binding sites within protein primary sequences to identify sites within immunoglobulin chains that might mediate their association with BiP. Very few of the sequential heptapeptides in the heavy or light chain sequences were potential BiP binding sites. Analysis of the ability of synthetic heptapeptides corresponding to 24 potential sites in heavy chains to stimulate the ATPase activity of BiP indicated that at least half of them were authentic BiP binding sequences. These sequences were not confined to a single domain of the heavy chain but were distributed within both the VH and CH domains. Interestingly, when the BiP binding sequences were mapped onto the three-dimensional structure of the Fd antibody fragment, the majority involve residues that participate in contact sites between the heavy and light chains. Therefore, we suggest that in vivo BiP chaperones the folding and assembly of antibody molecules by binding to hydrophobic surface regions on the isolated immunoglobulin chains that subsequently participate in interchain contacts.
Adenosine Triphosphatases↗
Interaction of GroEL with a highly structured folding intermediate: iterative binding cycles do not involve unfolding.
The GroE proteins are molecular chaperones involved in protein folding. The general mechanism by which they facilitate folding is still enigmatic. One of the central open questions is the conformation of the GroEL-bound nonnative protein. Several suggestions have been made concerning the folding stage at which a protein can interact with GroEL. Furthermore, the possibility exists that binding of the nonnative protein to GroEL results in its unfolding. We have addressed these issues that are basic for understanding the GroE-mediated folding cycle by using folding intermediates of an Fab antibody fragment as molecular probes to define the binding properties of GroEL. We show that, in addition to binding to an early folding intermediate, GroEL is able to recognize and interact with a late quaternary-structured folding intermediate (Dc) without measurably unfolding it. Thus, the prerequisite for binding is not a certain folding stage of a nonnative protein. In contrast, general surface properties of nonnative proteins seem to be crucial for binding. Furthermore, unfolding of a highly structured intermediate does not necessarily occur upon binding to GroEL. Folding of Dc in the presence of GroEL and ATP involves cycles of binding and release. Because in this system no off-pathway reactions or kinetic traps are involved, a quantitative analysis of the reactivation kinetics observed is possible. Our results indicate that the association reaction of Dc and GroEL in the presence of ATP is rather slow, whereas in the absence of ATP association is several orders of magnitude more efficient. Therefore, it seems that ATP functions by inhibiting reassociation rather than promoting release of the bound substrate.
Chaperonin 60↗
Functional characterization of the higher plant chloroplast chaperonins.
The higher plant chloroplast chaperonins (ch-cpn60 and ch-cpn10) have been purified and their structural/functional properties examined. In all plants surveyed, both proteins were constitutively expressed, and only modest increases in their levels were detected upon heat shock. Like GroEL and GroES of Escherichia coli, the chloroplast chaperonins can physically interact with each other. The asymmetric complexes that form in the presence of ADP are "bullet-shaped" particles that likely consist of 1 mol each of ch-cpn60 and ch-cpn10. The purified ch-cpn60 is a functional molecular chaperone. Under "nonpermissive" conditions, where spontaneous folding was not observed, it was able to assist in the refolding of two different target proteins. In both cases, successful partitioning to the native state also required ATP hydrolysis and chaperonin 10. Surprisingly, however, the "double-domain" ch-cpn10, comprised of unique 21-kDa subunits, was not an obligatory co-chaperonin. Both GroES and a mammalian mitochondrial homolog were equally compatible with the ch-cpn60. Finally, the assisted-folding reaction mediated by the chloroplast chaperonins does not require K+ ions. Thus, the K(+)-dependent ATPase activity that is observed with other known groEL homologs is not a universal property of all chaperonin 60s.
Adenosine Diphosphate↗
Structural organization of procaryotic and eucaryotic Hsp90. Influence of divalent cations on structure and function.
Hsp90 is a very abundant molecular chaperone that apparently helps to protect cellular proteins from denaturation upon temperature upshift. The unusual ability of Hsp90 to function under conditions where other proteins unfold prompted us to investigate the stability and structural organization of Hsp90 itself. Both procaryotic and eucaryotic members of the Hsp90 family were found to have very similar physicochemical properties: (i) they are stable against thermal unfolding up to at least 50 degrees C, (ii) they show biphasic, reversible unfolding transitions in guanidinium chloride, and (iii) their oligomerization state is strongly and rapidly affected by millimolar concentrations of divalent cations. In the presence of MnCl2 and MgCl2 defined changes in the quaternary structure of Hsp90 could be observed which resulted in a decrease in thermostability and an increased tendency to form larger aggregates. The addition of divalent cations also almost completely abolished the chaperone function of Hsp90 and induced release of folding intermediates of citrate synthase bound to Hsp90. These modulating effects of divalent cations on structure and function of Hsp90 in vitro represent a potential mechanism for regulation of Hsp90 chaperone action in vivo.
Animals↗
Association of antibody chains at different stages of folding: prolyl isomerization occurs after formation of quaternary structure.
The folding pathways of multi-domain proteins are still poorly understood due to the complexity of the reaction involving domain folding, association and, in many cases, prolyl cis/trans isomerization. Here, we have established a kinetic model for the folding of the Fab fragment of the antibody MAK 33 with intact disulfide bonds. Folding of the hetero-dimeric protein from the completely denatured, oxidized state comprises the pairwise association of the two domains of each chain with those of the partner protein. Both the reactivation of the Fab fragment in which the two constituent polypeptide chains were covalently linked via a cystine bond (Fab) and that of a mutant lacking this covalent linkage (Fab/-cys) were monitored by ELISA. Folding of the Fab fragment is a slow process, which can be described by a single exponential term. The kinetic phase reflects a folding step after the association of the two chains. The same reaction was detected in the folding of Fab/-cys but an additional rate-limiting step is involved that is due to a unimolecular step in the folding of the isolated light chain. This implies that, during Fab reactivation, Fd associates with the light chain at the stage of an earlier folding intermediate, thus eliminating the additional slow folding step of the light chain observed with Fab/-cys. Both in Fab and Fab/-cys renaturation, the folding reaction after association is determined by prolyl isomerization. Therefore, at least four different association-competent folding intermediates have to be postulated according to the folding stage of light chain and the configuration of at least one prolyl-peptide bond. Using the different substrate specificities of cyclophilin and FK506 binding protein, we have obtained evidence that Pro159 within the Fd fragment may be responsible for the observed slow folding phase after association, although three other proline residues adopt a cis configuration in the native protein. Furthermore, the data suggest that in the case of the Fab fragment, association is a prerequisite for cis/trans isomerization of prolyl peptide bonds, implying that the quaternary but not the tertiary structure determines the cis-configuration of the prolyl residue in Fd involved in the rate-limiting folding reaction.
Amino Acid Sequence↗
Transient interaction of Hsp90 with early unfolding intermediates of citrate synthase. Implications for heat shock in vivo.
At normal temperatures, Hsp90 is one of the most abundant proteins in the cytosol of various eucaryotic cells. Upon heat shock, the level of Hsp90 is increased even more, suggesting that it is important for helping cells to survive under these conditions. However, studies so far have been almost exclusively concerned with the function of Hsp90 under non-stress conditions, and therefore only little is known about the role of Hsp90 during heat shock. As a model for heat shock in vitro, we have monitored the inactivation and subsequent aggregation of dimeric citrate synthase (CS) at elevated temperatures. Hsp90 effectively "stabilized" CS under conditions where the enzyme is normally inactivated and finally aggregates very rapidly. A kinetic dissection of the unfolding pathway of CS succeeded in revealing two intermediates which form and subsequently undergo irreversible aggregation reactions. Hsp90 apparently interacts transiently with these highly structured early unfolding intermediates. Binding and subsequent release of the intermediates favorably influences the kinetic partitioning between two competing processes, the further unfolding of CS and the productive refolding to the native state. As a consequence, CS is effectively stabilized in the presence of Hsp90. The significance of this interaction is especially evident in the suppression of aggregation, the major end result of thermal unfolding events in vivo and in vitro. These effects, which are ATP-independent, seem to be a general function of members of the Hsp90 family, since yeast and bovine Hsp90 as well as the Hsp90 homologue from Escherichia coli gave similar results. It seems likely that this function also reflects the role of Hsp90 under heat shock conditions in vivo. We therefore propose that members of the Hsp90 family convey thermotolerance by transiently binding to highly structured early unfolding intermediates, thereby preventing their irreversible aggregation and stabilizing the active species.
Animals↗