Structural basis for the cooperation of Hsp70 and Hsp110 chaperones in protein folding

Protein folding by Hsp70 is tightly controlled by cochaperones, including J-domain proteins that trigger ATP hydrolysis and nucleotide exchange factors (NEFs) that remove ADP from Hsp70. Here we present the crystal structure of the yeast NEF Sse1p (Hsp110) in complex with the nucleotide-binding domain (NBD) of Hsp70. Hsp110 proteins are homologous to Hsp70s and consist of an NBD, a beta sandwich domain, and a three helix bundle domain (3HBD). In the complex, the NBD of Sse1p is ATP bound, and together with the 3HBD it embraces the NBD of Hsp70, inducing opening and the release of bound ADP from Hsp70. Mutations that abolish NEF activity are lethal, thus defining nucleotide exchange on Hsp70 as an essential function of Sse1p. Our data suggest that Sse1p does not employ the nucleotide-dependent allostery and peptide-binding mode of canonical Hsp70s, and that direct interactions of substrate with Sse1p may support Hsp70-assisted protein folding in a cooperative process.     

Hsp110 is a nucleotide-activated exchange factor for Hsp70

Hsp110 proteins constitute a subfamily of the Hsp70 chaperones and are potent nucleotide exchange factors (NEFs) for canonical Hsp70s of the eukaryotic cytosol. Here, we show that the NEF activity of the yeast Hsp110 homologue Sse1 itself is controlled by nucleotide. Nucleotide binding results in formation of a stabilized conformation of Sse1 that is required for association with the yeast Hsp70 Ssa1. The interaction triggers release of bound ADP from Ssa1, but nucleotide persists bound to Sse1 in the complex. Surprisingly, removal of this nucleotide does not affect the integrity of the complex. Instead, rebinding of ATP to the Hsp70 prompts the dissociation of the complex. Our data demonstrate that in contrast to previously characterized NEFs for Hsp70 chaperones, the NEF activity of Sse1 requires nucleotide binding and let us propose a new model for Hsp110 function.     

Molecular chaperones of the Hsp110 family act as nucleotide exchange factors of Hsp70s

Hsp70 molecular chaperones function in protein folding in a manner dependent on regulation by co-chaperones. Hsp40s increase the low intrinsic ATPase activity of Hsp70, and nucleotide exchange factors (NEFs) remove ADP after ATP hydrolysis, enabling a new Hsp70 interaction cycle with non-native protein substrate. Here, we show that members of the Hsp70-related Hsp110 family cooperate with Hsp70 in protein folding in the eukaryotic cytosol. Mammalian Hsp110 and the yeast homologues Sse1p/2p catalyze efficient nucleotide exchange on Hsp70 and its orthologue in Saccharomyces cerevisiae, Ssa1p, respectively. Moreover, Sse1p has the same effect on Ssb1p, a ribosome-associated isoform of Hsp70 in yeast. Mutational analysis revealed that the N-terminal ATPase domain and the ultimate C-terminus of Sse1p are required for nucleotide exchange activity. The Hsp110 homologues significantly increase the rate and yield of Hsp70-mediated re-folding of thermally denatured firefly luciferase in vitro. Similarly, deletion of SSE1 causes a firefly luciferase folding defect in yeast cells under heat stress in vivo. Our data indicate that Hsp110 proteins are important components of the eukaryotic Hsp70 machinery of protein folding.     

Structural basis of J cochaperone binding and regulation of Hsp70

The many protein processing reactions of the ATP-hydrolyzing Hsp70s are regulated by J cochaperones, which contain J domains that stimulate Hsp70 ATPase activity and accessory domains that present protein substrates to Hsp70s. We report the structure of a J domain complexed with a J responsive portion of a mammalian Hsp70. The J domain activates ATPase activity by directing the linker that connects the Hsp70 nucleotide binding domain (NBD) and substrate binding domain (SBD) toward a hydrophobic patch on the NBD surface. Binding of the J domain to Hsp70 displaces the SBD from the NBD, which may allow the SBD flexibility to capture diverse substrates. Unlike prokaryotic Hsp70, the SBD and NBD of the mammalian chaperone interact in the ADP state. Thus, although both nucleotides and J cochaperones modulate Hsp70 NBD:linker and NBD:SBD interactions, the intrinsic persistence of those interactions differs in different Hsp70s and this may optimize their activities for different cellular roles.     

Role of Hsp70 ATPase Domain Intrinsic Dynamics and Sequence Evolution in Enabling its Functional Interactions with NEFs

Catalysis of ADP-ATP exchange by nucleotide exchange factors (NEFs) is central to the activity of Hsp70 molecular chaperones. Yet, the mechanism of interaction of this family of chaperones with NEFs is not well understood in the context of the sequence evolution and structural dynamics of Hsp70 ATPase domains. We studied the interactions of Hsp70 ATPase domains with four different NEFs on the basis of the evolutionary trace and co-evolution of the ATPase domain sequence, combined with elastic network modeling of the collective dynamics of the complexes. Our study reveals a subtle balance between the intrinsic (to the ATPase domain) and specific (to interactions with NEFs) mechanisms shared by the four complexes. Two classes of key residues are distinguished in the Hsp70 ATPase domain: (i) highly conserved residues, involved in nucleotide binding, which mediate, via a global hinge-bending, the ATPase domain opening irrespective of NEF binding, and (ii) not-conserved but co-evolved and highly mobile residues, engaged in specific interactions with NEFs (e.g., N57, R258, R262, E283, D285). The observed interplay between these respective intrinsic (pre-existing, structure-encoded) and specific (co-evolved, sequence-dependent) interactions provides us with insights into the allosteric dynamics and functional evolution of the modular Hsp70 ATPase domain.     

Binding of Human Nucleotide Exchange Factors to Heat Shock Protein 70 (Hsp70) Generates Functionally Distinct Complexes in Vitro

Proteins with Bcl2-associated anthanogene (BAG) domains act as nucleotide exchange factors (NEFs) for the molecular chaperone heat shock protein 70 (Hsp70). There are six BAG family NEFs in humans, and each is thought to link Hsp70 to a distinct cellular pathway. However, little is known about how the NEFs compete for binding to Hsp70 or how they might differentially shape its biochemical activities. Toward these questions, we measured the binding of human Hsp72 (HSPA1A) to BAG1, BAG2, BAG3, and the unrelated NEF Hsp105. These studies revealed a clear hierarchy of affinities: BAG3 > BAG1 > Hsp105 ≫ BAG2. All of the NEFs competed for binding to Hsp70, and their relative affinity values predicted their potency in nucleotide and peptide release assays. Finally, we combined the Hsp70-NEF pairs with cochaperones of the J protein family (DnaJA1, DnaJA2, DnaJB1, and DnaJB4) to generate 16 permutations. The activity of the combinations in ATPase and luciferase refolding assays were dependent on the identity and stoichiometry of both the J protein and NEF so that some combinations were potent chaperones, whereas others were inactive. Given the number and diversity of cochaperones in mammals, it is likely that combinatorial assembly could generate a large number of distinct permutations.     

A lysine-rich region within fungal BAG domain-containing proteins mediates a novel association with ribosomes

Heat shock protein 70 (Hsp70) is a highly conserved molecular chaperone that assists in the folding of nascent chains and the repair of unfolded proteins through iterative cycles of ATP binding, hydrolysis, and nucleotide exchange tightly coupled to polypeptide binding and release. Cochaperones, including nucleotide exchange factors (NEFs), modulate the rate of ADP/ATP exchange and serve to recruit Hsp70 to distinct processes or locations. Among three nonrelated cytosolic NEFs in Saccharomyces cerevisiae, the Bag-1 homolog SNL1 is unique in being tethered to the endoplasmic reticulum (ER) membrane. We demonstrate here a novel physical association between Snl1 and the intact ribosome. This interaction is both independent of and concurrent with binding to Hsp70 and is not dependent on membrane localization. The ribosome binding site is identified as a short lysine-rich motif within the amino terminus of the Snl1 BAG domain distinct from the Hsp70 interaction region. Additionally, we demonstrate a ribosome association with the Candida albicans Snl1 homolog and localize this putative NEF to a perinuclear/ER membrane, suggesting functional conservation in fungal BAG domain-containing proteins. We therefore propose that the Snl1 family of NEFs serves a previously unknown role in fungal protein biogenesis based on the coincident recruitment of ribosomes and Hsp70 to the ER membrane.     

Overexpression,Purification and Characterisation of the Plasmodium falciparum Hsp70-z (PfHsp70-z) Protein

Six Hsp70-like genes are represented on the genome of Plasmodium falciparum. Of these two occur in the cytosol: P. falciparum Hsp70-z (PfHsp70-z) and PfHsp70-1. PfHsp70-1 is a well characterised canonical Hsp70 that facilitates protein quality control and is crucial for the development of malaria parasites. There is very little known about PfHsp70-z. However, PfHsp70-z is known to be essential and is implicated in suppressing aggregation of asparagine-rich proteins of P. falciparum. In addition, its expression at the clinical stage of malaria correlates with disease prognosis. Based on structural evidence PfHsp70-z belongs to the Hsp110 family of proteins. Since Hsp110 proteins have been described as nucleotide exchange factors (NEFs) of their canonical Hsp70 counterparts, it has been speculated that PfHsp70-z may serve as a NEF of PfHsp70-1. In the current study, P. falciparum cells cultured in vitro were subjected to heat stress, triggering the enhanced expression of PfHsp70-z. Biochemical assays conducted using recombinant PfHsp70-z protein demonstrated that the protein is heat stable and possesses ATPase activity. Furthermore, we observed that PfHsp70-z is capable of self-association. The structural-functional features of PfHsp70-z provide further evidence for its role as a chaperone and possible nucleotide exchange factor of PfHsp70-1.     

Molecular Mechanism of Allosteric Communication in Hsp70 Revealed by Molecular Dynamics Simulations

Investigating ligand-regulated allosteric coupling between protein domains is fundamental to understand cell-life regulation. The Hsp70 family of chaperones represents an example of proteins in which ATP binding and hydrolysis at the Nucleotide Binding Domain (NBD) modulate substrate recognition at the Substrate Binding Domain (SBD). Herein, a comparative analysis of an allosteric (Hsp70-DnaK) and a non-allosteric structural homolog (Hsp110-Sse1) of the Hsp70 family is carried out through molecular dynamics simulations, starting from different conformations and ligand-states. Analysis of ligand-dependent modulation of internal fluctuations and local deformation patterns highlights the structural and dynamical changes occurring at residue level upon ATP-ADP exchange, which are connected to the conformational transition between closed and open structures. By identifying the dynamically responsive protein regions and specific cross-domain hydrogen-bonding patterns that differentiate Hsp70 from Hsp110 as a function of the nucleotide, we propose a molecular mechanism for the allosteric signal propagation of the ATP-encoded conformational signal.     

Characterization of Hsp70 binding and nucleotide exchange by the yeast Hsp110 chaperone Sse1

SSE1 and SSE2 encode the essential yeast members of the Hsp70-related Hsp110 molecular chaperone family. Both mammalian Hsp110 and the Sse proteins functionally interact with cognate cytosolic Hsp70s as nucleotide exchange factors. We demonstrate here that Sse1 forms high-affinity (Kd approximately 10-8 M) heterodimeric complexes with both yeast Ssa and mammalian Hsp70 chaperones and that binding of ATP to Sse1 is required for binding to Hsp70s. Sse1.Hsp70 heterodimerization confers resistance to exogenously added protease, indicative of conformational changes in Sse1 resulting in a more compact structure. The nucleotide binding domains of both Sse1/2 and the Hsp70s dictate interaction specificity and are sufficient for mediating heterodimerization with no discernible contribution from the peptide binding domains. In support of a strongly conserved functional interaction between Hsp110 and Hsp70, Sse1 is shown to associate with and promote nucleotide exchange on human Hsp70. Nucleotide exchange activity by Sse1 is physiologically significant, as deletion of both SSE1 and the Ssa ATPase stimulatory protein YDJ1 is synthetically lethal. The Hsp110 family must therefore be considered an essential component of Hsp70 chaperone biology in the eukaryotic cell.     

<Emphasis Type="Italic">Plasmodium falciparum</Emphasis> Hsp70-z,an Hsp110 homologue,exhibits independent chaperone activity and interacts with Hsp70-1 in a nucleotide-dependent fashion

The role of molecular chaperones, among them heat shock proteins (Hsps), in the development of malaria parasites has been well documented. Hsp70s are molecular chaperones that facilitate protein folding. Hsp70 proteins are composed of an N-terminal nucleotide binding domain (NBD), which confers them with ATPase activity and a C-terminal substrate binding domain (SBD). In the ADP-bound state, Hsp70 possesses high affinity for substrate and releases the folded substrate when it is bound to ATP. The two domains are connected by a conserved linker segment. Hsp110 proteins possess an extended lid segment, a feature that distinguishes them from canonical Hsp70s. Plasmodium falciparum Hsp70-z (PfHsp70-z) is a member of the Hsp110 family of Hsp70-like proteins. PfHsp70-z is essential for survival of malaria parasites and is thought to play an important role as a molecular chaperone and nucleotide exchange factor of its cytosolic canonical Hsp70 counterpart, PfHsp70-1. Unlike PfHsp70-1 whose functions are fairly well established, the structure-function features of PfHsp70-z remain to be fully elucidated. In the current study, we established that PfHsp70-z possesses independent chaperone activity. In fact, PfHsp70-z appears to be marginally more effective in suppressing protein aggregation than its cytosol-localized partner, PfHsp70-1. Furthermore, based on coimmunoaffinity chromatography and surface plasmon resonance analyses, PfHsp70-z associated with PfHsp70-1 in a nucleotide-dependent fashion. Our findings suggest that besides serving as a molecular chaperone, PfHsp70-z could facilitate the nucleotide exchange function of PfHsp70-1. These dual functions explain why it is essential for parasite survival.     

Crystal structure of the nucleotide‐binding domain of mortalin,the mitochondrial Hsp70 chaperone

Mortalin, a member of the Hsp70‐family of molecular chaperones, functions in a variety of processes including mitochondrial protein import and quality control, Fe‐S cluster protein biogenesis, mitochondrial homeostasis, and regulation of p53. Mortalin is implicated in regulation of apoptosis, cell stress response, neurodegeneration, and cancer and is a target of the antitumor compound MKT‐077. Like other Hsp70‐family members, Mortalin consists of a nucleotide‐binding domain (NBD) and a substrate‐binding domain. We determined the crystal structure of the NBD of human Mortalin at 2.8 Å resolution. Although the Mortalin nucleotide‐binding pocket is highly conserved relative to other Hsp70 family members, we find that its nucleotide affinity is weaker than that of Hsc70. A Parkinson's disease‐associated mutation is located on the Mortalin‐NBD surface and may contribute to Mortalin aggregation. We present structure‐based models for how the Mortalin‐NBD may interact with the nucleotide exchange factor GrpEL1, with p53, and with MKT‐077. Our structure may contribute to the understanding of disease‐associated Mortalin mutations and to improved Mortalin‐targeting antitumor compounds.     

Functional divergence between co-chaperones of Hsc70

The ATPase cycle of the chaperone Hsc70 is regulated by co-chaperones; Hsp40/DnaJ-related proteins stimulate ATP hydrolysis by Hsc70 and can bind unfolded polypeptides themselves. Conversely, various nucleotide exchange factors (NEFs) stimulate ADP-ATP exchange by Hsc70. We analyzed the purified Hsp40-related co-chaperones DJA1 (Hdj2) and DJA2 (Hdj3) and found that they had a distinct pattern of binding to a range of polypeptides. DJA2 alone could stimulate Hsc70-mediated refolding of luciferase in the absence of NEF, whereas DJA1 was much less active. The addition of the Bag1 NEF increased refolding by Hsc70 and DJA2, as did the newly characterized NEF Hsp110, but each NEF had a different optimal concentration ratio to Hsc70. Notably, the NEF HspBP1 could not increase refolding by Hsc70 and DJA2 at any concentration, and none of the NEFs improved the refolding activity with DJA1. Instead, DJA1 was inhibitory of refolding with DJA2 and Hsc70. All combinations of DJA1 or DJA2 with the three NEFs stimulated the Hsc70 ATPase rate, although Hsp110 became less effective with increasing concentrations. A chimeric DJA2 having its Hsc70-stimulatory J domain replaced with that of DJA1 was functional for polypeptide binding and ATPase stimulation of Hsc70. However, it could not support efficient Hsc70-mediated refolding and also inhibited refolding with DJA2 and Hsc70. These results suggest a more complex model of Hsc70 mechanism than has been previously thought, with notable functional divergence between Hsc70 co-chaperones.     

Allostery in Hsp70 Chaperones Is Transduced by Subdomain Rotations

Hsp70s (heat shock protein 70 kDa) are central to protein folding, refolding, and trafficking in organisms ranging from archaea to Homo sapiens under both normal and stressed cellular conditions. Hsp70s are comprised of a nucleotide-binding domain (NBD) and a substrate-binding domain (SBD). The nucleotide binding site in the NBD and the substrate binding site in the SBD are allosterically linked: ADP binding promotes substrate binding, while ATP binding promotes substrate release. Hsp70s have been linked to inhibition of apoptosis (i.e., cancer) and diseases associated with protein misfolding such as Alzheimer's, Parkinson's, and Huntington's.It has long been a goal to characterize the nature of allosteric coupling in these proteins. However, earlier studies of the isolated NBD could not show any difference in overall conformation between the ATP state and the ADP state. Hence the question: How is the state of the nucleotide communicated between NBD and SBD?Here we report a solution NMR study of the 44-kDa NBD of Hsp70 from Thermus thermophilus in the ADP and AMPPNP states. Using the solution NMR methods of residual dipolar coupling analysis, we determine that significant rotations occur for different subdomains of the NBD upon exchange of nucleotide. These rotations modulate access to the nucleotide binding cleft in the absence of a nucleotide exchange factor. Moreover, the rotations cause a change in the accessibility of a hydrophobic surface cleft remote from the nucleotide binding site, which previously has been identified as essential to allosteric communication between NBD and SBD. We propose that it is this change in the NBD surface cleft that constitutes the allosteric signal that can be recognized by the SBD.     

Regulation of Human Hsc70 ATPase and Chaperone Activities by Apg2: Role of the Acidic Subdomain

Protein aggregate reactivation in metazoans is accomplished by the combined activity of Hsp70, Hsp40 and Hsp110 chaperones. Hsp110s support the refolding of aggregated polypeptides acting as specialized nucleotide exchange factors of Hsp70. We have studied how Apg2, one of the three human Hsp110s, regulates the activity of Hsc70 (HspA8), the constitutive Hsp70 in our cells. Apg2 shows a biphasic behavior: at low concentration, it stimulates the ATPase cycle of Hsc70, binding of the chaperone to protein aggregates and the refolding activity of the system, while it inhibits these three processes at high concentration. When the acidic subdomain of Apg2, a characteristic sequence present in the substrate binding domain of all Hsp110s, is deleted, the detrimental effects occur at lower concentration and are more pronounced, which concurs with an increase in the affinity of the Apg2 mutant for Hsc70. Our data support a mechanism in which Apg2 arrests the chaperone cycle through an interaction with Hsc70(ATP) that might lead to premature ATP dissociation before hydrolysis. In this line, the acidic subdomain might serve as a conformational switch to support dissociation of the Hsc70:Apg2 complex.     

Structural basis of interdomain communication in the Hsc70 chaperone

Hsp70 family proteins are highly conserved chaperones involved in protein folding, degradation, targeting and translocation, and protein complex remodeling. They are comprised of an N-terminal nucleotide binding domain (NBD) and a C-terminal protein substrate binding domain (SBD). ATP binding to the NBD alters SBD conformation and substrate binding kinetics, but an understanding of the mechanism of interdomain communication has been hampered by the lack of a crystal structure of an intact chaperone. We report here the 2.6 angstroms structure of a functionally intact bovine Hsc70 (bHsc70) and a mutational analysis of the observed interdomain interface and the immediately adjacent interdomain linker. This analysis identifies interdomain interactions critical for chaperone function and supports an allosteric mechanism in which the interdomain linker invades and disrupts the interdomain interface when ATP binds.     

Common functionally important motions of the nucleotide‐binding domain of Hsp70

The 70 kDa heat shock proteins (Hsp70) are a family of molecular chaperones involved in protein folding, aggregate prevention, and protein disaggregation. They consist of the substrate‐binding domain (SBD) that binds client substrates, and the nucleotide‐binding domain (NBD), whose cycles of nucleotide hydrolysis and exchange underpin the activity of the chaperone. To characterize the structure–function relationships that link the binding state of the NBD to its conformational behavior, we analyzed the dynamics of the NBD of the Hsp70 chaperone from Bos taurus (PDB 3C7N:B) by all‐atom canonical molecular dynamics simulations. It was found that essential motions within the NBD fall into three major classes: the mutual class, reflecting tendencies common to all binding states, and the ADP‐ and ATP‐unique classes, which reflect conformational trends that are unique to either the ADP‐ or ATP‐bound states, respectively. “Mutual” class motions generally describe “in‐plane” and/or “out‐of‐plane” (scissor‐like) rotation of the subdomains within the NBD. This result is consistent with experimental nuclear magnetic resonance data on the NBD. The “unique” class motions target specific regions on the NBD, usually surface loops or sites involved in nucleotide binding and are, therefore, expected to be involved in allostery and signal transmission. For all classes, and especially for those of the “unique” type, regions of enhanced mobility can be identified; these are termed “hot spots,” and their locations generally parallel those found by NMR spectroscopy. The presence of magnesium and potassium cations in the nucleotide‐binding pocket was also found to influence the dynamics of the NBD significantly. Proteins 2015; 83:282–299. © 2014 Wiley Periodicals, Inc.     

Swapping nucleotides, tuning Hsp70

Molecular chaperones such as heat shock protein 70 (Hsp70) are crucial for protein folding. Crystal structures of Hsp70 in a complex with the nucleotide exchange factor (NEF) Hsp110 reported in this issue of Cell (Polier et al., 2008) and in Molecular Cell (Schuermann et al., 2008) provide new insights into how NEF action specifies Hsp70 cellular function.     

Allosteric communication between the nucleotide binding domains of caseinolytic peptidase B

ClpB is a hexameric chaperone that solubilizes and reactivates protein aggregates in cooperation with the Hsp70/DnaK chaperone system. Each of the identical protein monomers contains two nucleotide binding domains (NBD), whose ATPase activity must be coupled to exert on the substrate the mechanical work required for its reactivation. However, how communication between these sites occurs is at present poorly understood. We have studied herein the affinity of each of the NBDs for nucleotides in WT ClpB and protein variants in which one or both sites are mutated to selectively impair nucleotide binding or hydrolysis. Our data show that the affinity of NBD2 for nucleotides (K(d) = 3-7 μm) is significantly higher than that of NBD1. Interestingly, the affinity of NBD1 depends on nucleotide binding to NBD2. Binding of ATP, but not ADP, to NBD2 increases the affinity of NBD1 (the K(d) decreases from ≈160-300 to 50-60 μm) for the corresponding nucleotide. Moreover, filling of the NBD2 ring with ATP allows the cooperative binding of this nucleotide and substrates to the NBD1 ring. Data also suggest that a minimum of four subunits cooperate to bind and reactivate two different aggregated protein substrates.     

Insights into Hsp70 chaperone activity from a crystal structure of the yeast Hsp110 Sse1

Classic Hsp70 chaperones assist in diverse processes of protein folding and translocation, and Hsp110s had seemed by sequence to be distant relatives within an Hsp70 superfamily. The 2.4 A resolution structure of Sse1 with ATP shows that Hsp110s are indeed Hsp70 relatives, and it provides insight into allosteric coupling between sites for ATP and polypeptide-substrate binding in Hsp70s. Subdomain structures are similar in intact Sse1(ATP) and in the separate Hsp70 domains, but conformational dispositions are radically different. Interfaces between Sse1 domains are extensive, intimate, and conservative in sequence with Hsp70s. We propose that Sse1(ATP) may be an evolutionary vestige of the Hsp70(ATP) state, and an analysis of 64 mutant variants in Sse1 and three Hsp70 homologs supports this hypothesis. An atomic-level understanding of Hsp70 communication between ATP and substrate-binding domains follows. Requirements on Sse1 for yeast viability are in keeping with the distinct function of Hsp110s as nucleotide exchange factors.