Disruption of Ionic Interactions between the Nucleotide Binding Domain 1 (NBD1) and Middle (M) Domain in Hsp100 Disaggregase Unleashes Toxic Hyperactivity and Partial Independence from Hsp70

Hsp100 chaperones cooperate with the Hsp70 chaperone system to disaggregate and reactivate heat-denatured aggregated proteins to promote cell survival after heat stress. The homology models of Hsp100 disaggregases suggest the presence of a conserved network of ionic interactions between the first nucleotide binding domain (NBD1) and the coiled-coil middle subdomain, the signature domain of disaggregating chaperones. Mutations intended to disrupt the putative ionic interactions in yeast Hsp104 and bacterial ClpB disaggregases resulted in remarkable changes of their biochemical properties. These included an increase in ATPase activity, a significant increase in the rate of in vitro substrate renaturation, and partial independence from the Hsp70 chaperone in disaggregation. Paradoxically, the increased activities resulted in serious growth impediments in yeast and bacterial cells instead of improvement of their thermotolerance. Our results suggest that this toxic activity is due to the ability of the mutated disaggregases to unfold independently from Hsp70, native folded proteins. Complementary changes that restore particular salt bridges within the suggested network suppressed the toxic effects. We propose a novel structural aspect of Hsp100 chaperones crucial for specificity and efficiency of the disaggregation reaction.     

The ClpB/Hsp104 molecular chaperone-a protein disaggregating machine

ClpB and Hsp104 (ClpB/Hsp104) are essential proteins of the heat-shock response and belong to the class 1 family of Clp/Hsp100 AAA+ ATPases. Members of this family form large ring structures and contain two AAA+ modules, which consist of a RecA-like nucleotide-binding domain (NBD) and an alpha-helical domain. Furthermore, ClpB/Hsp104 has a longer middle region, the ClpB/Hsp104-linker, which is essential for chaperone activity. Unlike other Clp/Hsp100 proteins, however, ClpB/Hsp104 neither associates with a cellular protease nor directs the degradation of its substrate proteins. Rather, ClpB/Hsp104 is a bona fide molecular chaperone, which has the remarkable ability to rescue proteins from an aggregated state. The full recovery of these proteins requires the assistance of the cognate DnaK/Hsp70 chaperone system. The mechanism of this "bi-chaperone" network, however, remains elusive. Here we review the current understanding of the structure-function relationship of the ClpB/Hsp104 molecular chaperone and its role in protein disaggregation.     

Crystal structure of E. coli Hsp100 ClpB nucleotide-binding domain 1 (NBD1) and mechanistic studies on ClpB ATPase activity

E. coli Hsp100 ClpB was recently identified as a critical part in a multi-chaperone system to play important roles in protein folding, protein transport and degradation in cell physiology. ClpB contains two nucleotide-binding domains (NBD1 and NBD2) within their primary sequences. NBD1 and NBD2 of ClpB can be classified as members of the large ATPase family known as ATPases associated with various cellular activities (AAA). To investigate how ClpB performs its ATPase activities for its chaperone activity, we have determined the crystal structure of ClpB nucleotide-binding domain 1 (NBD1) by MAD method to 1.80 A resolution. The NBD1 monomer structure contains one domain that comprises 11 alpha-helices and six beta-strands. When compared with the typical AAA structures, the crystal structure of ClpB NBD1 reveals a novel AAA topology with six-stranded beta-sheet as its core. The N-terminal portion of NBD1 structure has an extra beta-strand flanked by two extra alpha-helices that are not present in other AAA structures. Moreover, the NBD1 structure does not have a C-terminal helical domain as other AAA proteins do. No nucleotide molecule is bound with ClpB NBD1 in the crystal structure probably due to lack of the C-terminal helix domain in the structure. Isothermal titration calorimetry (ITC) studies of ClpB NBD1 and other ClpB deletion mutations showed that either ClpB NBD1 or NBD2 alone does not bind to nucleotides. However, ClpB NBD2 combined with ClpB C-terminal fragment can interact with one ADP or ATP molecule. ITC data also indicated that full-length ClpB could bind two ADP molecules or one ATP analogue ATPgammaS molecule. Further ATPase activity studies of ClpB and ClpB deletion mutants showed that only wild-type ClpB have ATPase activity. None of ClpB NBD1 domain, NBD2 domain and NBD2 with C-terminal fragment has detectable ATPase activities. On the basis of our structural and mutagenesis data, we proposed a "see-saw" model to illustrate the mechanisms by which ClpB performs its ATPase activities for chaperone functions.     

Hsp101 is necessary for heat tolerance but dispensable for development and germination in the absence of stress

Hsp101 is a molecular chaperone that is required for the development of thermotolerance in plants and other organisms. We report that Arabidopsis thaliana Hsp101 is also regulated during seed development in the absence of stress, in a pattern similar to that seen for LEA proteins and small Hsps; protein accumulates during mid-maturation and is stored in the dry seed. Two new alleles of the locus encoding Hsp101 (HOT1) were isolated from Arabidopsis T-DNA mutant populations. One allele, hot1-3, contains an insertion within the second exon and is null for Hsp101 protein expression. Despite the complete absence of Hsp101 protein, plant growth and development, as well as seed germination, are normal, demonstrating that Hsp101 chaperone activity is not essential in the absence of stress. In thermotolerance assays hot1-3 shows a similar, though somewhat more severe, phenotype to the previously described missense allele hot1-1, revealing that the hot1-1 mutation is also close to null for protein activity. The second new mutant allele, hot1-2, has an insertion in the promoter 101 bp 5' to the putative TATA element. During heat stress the hot1-2 mutant produces normal levels of protein in hypocotyls and 10-day-old seedlings, and it is wild type for thermotolerance at these stages. Thus this mutation has not disrupted the minimal promoter sequence required for heat regulation of Hsp101. The hot1-2 mutant also expresses Hsp101 in seeds, but at a tenfold reduced level, resulting in reduced thermotolerance of germinating seeds and underscoring the importance of Hsp101 to seed stress tolerance.     

The Molecular Mechanism of Hsp100 Chaperone Inhibition by the Prion Curing Agent Guanidinium Chloride

The Hsp100 chaperones ClpB and Hsp104 utilize the energy from ATP hydrolysis to reactivate aggregated proteins in concert with the DnaK/Hsp70 chaperone system, thereby playing an important role in protein quality control. They belong to the family of AAA+ proteins (ATPases associated with various cellular activities), possess two nucleotide binding domains per monomer (NBD1 and NBD2), and oligomerize into hexameric ring complexes. Furthermore, Hsp104 is involved in yeast prion propagation and inheritance. It is well established that low concentrations of guanidinium chloride (GdmCl) inhibit the ATPase activity of Hsp104, leading to so called “prion curing,” the loss of prion-related phenotypes. Here, we present mechanistic details about the Hsp100 chaperone inhibition by GdmCl using the Hsp104 homolog ClpB from Thermus thermophilus. Initially, we demonstrate that NBD1 of ClpB, which was previously considered inactive as a separately expressed construct, is a fully active ATPase on its own. Next, we show that only NBD1, but not NBD2, is affected by GdmCl. We present a crystal structure of ClpB NBD1 in complex with GdmCl and ADP, showing that the Gdm⁺ ion binds specifically to the active site of NBD1. A conserved essential glutamate residue is involved in this interaction. Additionally, Gdm⁺ interacts directly with the nucleotide, thereby increasing the nucleotide binding affinity of NBD1. We propose that both the interference with the essential glutamate and the modulation of nucleotide binding properties in NBD1 is responsible for the GdmCl-specific inhibition of Hsp100 chaperones.     

ATP binding to nucleotide binding domain (NBD)1 of the ClpB chaperone induces motion of the long coiled-coil, stabilizes the hexamer, and activates NBD2

The molecular chaperone ClpB can rescue the heat-damaged proteins from an aggregated state in cooperation with other chaperones. It has two nucleotide binding domains (NBD1 and NBD2) and forms a hexamer ring in a manner dependent on ATP binding to NBD1. In the crystal structure of ClpB with both NBDs filled by nucleotides, the linker between two NBDs forms an 85-A-long coiled-coil that extends on the outside of the hexamer and leans to NBD1. To probe the possible motion of the coiled-coil, we tested the accessibility of a labeling reagent, fluorescence change of a labeled dye, and cross-linking between the coiled-coil and NBD1 by using the mutants with defective NBD1 or NBD2. The results suggest that the coiled-coil is more or less parallel to the main body of ClpB in the absence of nucleotide and that ATP binding to NBD1 brings it to the leaning position as seen in the crystal structure. This motion results in stabilization of the hexamer form of ClpB and promotion of ATP hydrolysis at NBD2.     

The elusive middle domain of Hsp104 and ClpB: location and function

Hsp104 in yeast and ClpB in bacteria are homologous, hexameric AAA+ proteins and Hsp100 chaperones, which function in the stress response as ring-translocases that drive protein disaggregation and reactivation. Both Hsp104 and ClpB contain a distinctive coiled-coil middle domain (MD) inserted in the first AAA+ domain, which distinguishes them from other AAA+ proteins and Hsp100 family members. Here, we focus on recent developments concerning the location and function of the MD in these hexameric molecular machines, which remains an outstanding question. While the atomic structure of the hexameric assembly of Hsp104 and ClpB remains uncertain, recent advances have illuminated that the MD is critical for the intrinsic disaggregase activity of the hexamer and mediates key functional interactions with the Hsp70 chaperone system (Hsp70 and Hsp40) that empower protein disaggregation.     

Molecular chaperones: structure of a protein disaggregase

The ring-forming molecular chaperone Hsp104/ClpB is a member of the AAA+ protein family which rescues proteins from aggregated states. The newly determined crystal structure of ClpB provides new insights into the mechanism of protein disaggregation, suggesting a crowbar activity mediated by a unique coiled-coil domain.     

Structure and function of Hsp78, the mitochondrial ClpB homolog

The cellular role of Hsp100/Clp chaperones in maintaining protein stability is based on two functional aspects. Under normal growth conditions they represent components of cellular protein quality control machineries that selectively remove damaged or misfolded polypeptides in cooperation with specific proteases. After thermal stress, proteins of the ClpB subfamily have the unique ability to directly resolubilize aggregated polypeptides in concert with Hsp70-type chaperones, leading to the recovery of enzymatic activity. Hsp78, the homolog of the bacterial chaperone ClpB in mitochondria of eukaryotic organisms, participates in both protective activities. Hsp78 is involved in conferring thermotolerance to the mitochondrial compartment but also participates in protein degradation by the matrix protease Pim1. Despite the high sequence conservation between Hsp78 and ClpB, an analysis of the structural properties revealed significant differences. The identified mitochondrial Hsp78s do not contain N-terminal substrate-binding domains. In addition, formation of the oligomeric chaperone complex was more variable as anticipated from the studies with bacterial ClpB. Hsp78 predominantly formed a trimeric complex under in vivo conditions. Hence, mitochondrial Hsp78s form a distinct subgroup of the ClpB chaperone family, exhibiting specific structural and functional properties.     

Conserved Distal Loop Residues in the Hsp104 and ClpB Middle Domain Contact Nucleotide-binding Domain 2 and Enable Hsp70-dependent Protein Disaggregation

The homologous hexameric AAA⁺ proteins, Hsp104 from yeast and ClpB from bacteria, collaborate with Hsp70 to dissolve disordered protein aggregates but employ distinct mechanisms of intersubunit collaboration. How Hsp104 and ClpB coordinate polypeptide handover with Hsp70 is not understood. Here, we define conserved distal loop residues between middle domain (MD) helix 1 and 2 that are unexpectedly critical for Hsp104 and ClpB collaboration with Hsp70. Surprisingly, the Hsp104 and ClpB MD distal loop does not contact Hsp70 but makes intrasubunit contacts with nucleotide-binding domain 2 (NBD2). Thus, the MD does not invariably project out into solution as in one structural model of Hsp104 and ClpB hexamers. These intrasubunit contacts as well as those between MD helix 2 and NBD1 are different in Hsp104 and ClpB. NBD2-MD contacts dampen disaggregase activity and must separate for protein disaggregation. We demonstrate that ClpB requires DnaK more stringently than Hsp104 requires Hsp70 for protein disaggregation. Thus, we reveal key differences in how Hsp104 and ClpB coordinate polypeptide handover with Hsp70, which likely reflects differential tuning for yeast and bacterial proteostasis.     

trans-Acting Arginine Residues in the AAA+ Chaperone ClpB Allosterically Regulate the Activity through Inter- and Intradomain Communication

The molecular chaperone ClpB/Hsp104, a member of the AAA+ superfamily (ATPases associated with various cellular activities), rescues proteins from the aggregated state in collaboration with the DnaK/Hsp70 chaperone system. ClpB/Hsp104 forms a hexameric, ring-shaped complex that functions as a tightly regulated, ATP-powered molecular disaggregation machine. Highly conserved and essential arginine residues, often called arginine fingers, are located at the subunit interfaces of the complex, which also harbor the catalytic sites. Several AAA+ proteins, including ClpB/Hsp104, possess a pair of such trans-acting arginines in the N-terminal nucleotide binding domain (NBD1), both of which were shown to be crucial for oligomerization and ATPase activity. Here, we present a mechanistic study elucidating the role of this conserved arginine pair. First, we found that the arginines couple nucleotide binding to oligomerization of NBD1, which is essential for the activity. Next, we designed a set of covalently linked, dimeric ClpB NBD1 variants, carrying single subunits deficient in either ATP binding or hydrolysis, to study allosteric regulation and intersubunit communication. Using this well defined environment of site-specifically modified, cross-linked AAA+ domains, we found that the conserved arginine pair mediates the cooperativity of ATP binding and hydrolysis in an allosteric fashion.     

Coupling and dynamics of subunits in the hexameric AAA+ chaperone ClpB

The bacterial AAA+ protein ClpB and its eukaryotic homologue Hsp104 ensure thermotolerance of their respective organisms by reactivating aggregated proteins in cooperation with the Hsp70/Hsp40 chaperone system. Like many members of the AAA+ superfamily, the ClpB protomers form ringlike homohexameric complexes. The mechanical energy necessary to disentangle protein aggregates is provided by ATP hydrolysis at the two nucleotide-binding domains of each monomer. Previous studies on ClpB and Hsp104 show a complex interplay of domains and subunits resulting in homotypic and heterotypic cooperativity. Using mutations in the Walker A and Walker B nucleotide-binding motifs in combination with mixing experiments we investigated the degree of inter-subunit coupling with respect to different aspects of the ClpB working cycle. We find that subunits are tightly coupled with regard to ATPase and chaperone activity, but no coupling can be observed for ADP binding. Comparison of the data with statistical calculations suggests that for double Walker mutants, approximately two in six subunits are sufficient to abolish chaperone and ATPase activity completely. In further experiments, we determined the dynamics of subunit reshuffling. Our results show that ClpB forms a very dynamic complex, reshuffling subunits on a timescale comparable to steady-state ATP hydrolysis. We propose that this could be a protection mechanism to prevent very stable aggregates from becoming suicide inhibitors for ClpB.     

Roles of the two ATP binding sites of ClpB from Thermus thermophilus.

As a member of molecular chaperone Hsp100/Clp family, TClpB from Thermus thermophilus has two nucleotide binding domains, NBD1 and NBD2, in a single polypeptide, each containing WalkerA and WalkerB consensus motifs. To probe their roles, mutations were introduced into the WalkerA or WalkerB motifs of each or both of the NBDs. The results are as follows. 1) For each of the NBDs, the ability of nucleotide binding is lost by mutations in the WalkerA motif but is retained by mutations in the WalkerB motif. 2) Each NBD has a casein-stimulatable small basic ATPase activity that is lost when the WalkerB motif is mutated. 3) TClpB assembles into a uniform 580-kDa oligomer when ATP is present at 55 degrees C, and only the mutants in the WalkerA motif in NBD1 fail to assemble, indicating that ATP binding to NBD1 but not hydrolysis is necessary and sufficient for the assembly. 4) Chaperone function of TClpB was lost when the WalkerA motif in each of the NBDs was mutated. Mutants in the WalkerB motifs of each NBD retained some chaperone activity.     

Cooperative kinetics of both Hsp104 ATPase domains and interdomain communication revealed by AAA sensor-1 mutants.

AAA proteins share a conserved active site for ATP hydrolysis and regulate many cellular processes. AAA proteins are oligomeric and often have multiple ATPase domains per monomer, which is suggestive of complex allosteric kinetics of ATP hydrolysis. Here, using wild-type Hsp104 in the hexameric state, we demonstrate that its two AAA modules (NBD1 and NBD2) have very different catalytic activities, but each displays cooperative kinetics of hydrolysis. Using mutations in the AAA sensor-1 motif of NBD1 and NBD2 that reduce the rate of ATP hydrolysis without affecting nucleotide binding, we also examine the consequences of keeping each site in the ATP-bound state. In vitro, reducing k(cat) at NBD2 significantly alters the steady-state kinetic behavior of NBD1. Thus, Hsp104 exhibits allosteric communication between the two sites in addition to homotypic cooperativity at both NBD1 and NBD2. In vivo, each sensor-1 mutation causes a loss-of-function phenotype in two assays of Hsp104 function (thermotolerance and yeast prion propagation), demonstrating the importance of ATP hydrolysis as distinct from ATP binding at each site for Hsp104 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.     

Role of a conserved aspartic acid in nucleotide binding domain 1 (NBD1) of Hsp100 chaperones in their activities

Besides its beneficial role in thermotolerance, the chaperone protein Hsp104 is involved in the inheritance of yeast Saccharomyces cerevisiae prions. Guanidine hydrochloride was previously shown to interfere with Hsp104 chaperone activity in vivo, thus impairing thermotolerance and resulting in prion curing. It was also reported that guanidine inhibits Hsp104 ATPase and disaggregation activity. We show that in vitro guanidine significantly inhibits the disaggregation activity of ClpB, the bacterial orthologue of Hsp104. However, guanidine exerts opposite effects on the ATPase activities of Hsp104 and ClpB. While the ATPase activity of Hsp104 is inhibited, the analogous ClpB activity is stimulated several-fold. Mutation of the universally conserved aspartic acid residue in position 184 to serine (D184S) in HSP104 and the analogous mutation in clpB (D178S) resulted in chaperones with lower disaggregating and ATPase activities. The activities of such changed chaperones are not influenced by guanidine, which suggests the role of this residue in the interaction with guanidine.     

Prokaryotic chaperones support yeast prions and thermotolerance and define disaggregation machinery interactions

Saccharomyces cerevisiae Hsp104 and Escherichia coli ClpB are Hsp100 family AAA+ chaperones that provide stress tolerance by cooperating with Hsp70 and Hsp40 to solubilize aggregated protein. Hsp104 also remodels amyloid in vitro and promotes propagation of amyloid prions in yeast, but ClpB does neither, leading to a view that Hsp104 evolved these activities. Although biochemical analyses identified disaggregation machinery components required for resolubilizing proteins, interactions among these components required for in vivo functions are not clearly defined. We express prokaryotic chaperones in yeast to address these issues and find ClpB supports both prion propagation and thermotolerance in yeast if it is modified to interact with yeast Hsp70 or if E. coli Hsp70 and its cognate nucleotide exchange factor (NEF) are present. Our findings show prion propagation and thermotolerance in yeast minimally require cooperation of species-specific Hsp100, Hsp70, and NEF with yeast Hsp40. The functions of this machinery in prion propagation were directed primarily by Hsp40 Sis1p, while thermotolerance relied mainly on Hsp40 Ydj1p. Our results define cooperative interactions among these components that are specific or interchangeable across life kingdoms and imply Hsp100 family disaggregases possess intrinsic amyloid remodeling activity.     

Cooperative action of Escherichia coli ClpB protein and DnaK chaperone in the activation of a replication initiation protein

The Escherichia coli molecular chaperone protein ClpB is a member of the highly conserved Hsp100/Clp protein family. Previous studies have shown that the ClpB protein is needed for bacterial thermotolerance. Purified ClpB protein has been shown to reactivate chemically and heat-denatured proteins. In this work we demonstrate that the combined action of ClpB and the DnaK, DnaJ, and GrpE chaperones leads to the activation of DNA replication of the broad-host-range plasmid RK2. In contrast, ClpB is not needed for the activation of the oriC-dependent replication of E. coli. Using purified protein components we show that the ClpB/DnaK/DnaJ/GrpE synergistic action activates the plasmid RK2 replication initiation protein TrfA by converting inactive dimers to an active monomer form. In contrast, Hsp78/Ssc1/Mdj1/Mge1, the corresponding protein system from yeast mitochondria, cannot activate the TrfA replication protein. Our results demonstrate for the first time that the ClpB/DnaK/DnaJ/GrpE system is involved in protein monomerization and in the activation of a DNA replication factor.     

A mutation in Thermosensitive Male Sterile 1, encoding a heat shock protein with DnaJ and PDI domains, leads to thermosensitive gametophytic male sterility in Arabidopsis

In most flowering plant species, pollination and fertilization occur during the hot summer, so plants must have evolved a mechanism that ensures normal growth of their pollen tubes at high temperatures. Despite its importance to plant reproduction, little is known about the molecular basis of thermotolerance in pollen tubes. Here we report the identification and characterization of a novel Arabidopsis gene, THERMOSENSITIVE MALE STERILE 1 ( TMS1 ), which plays an important role in thermotolerance of pollen tubes. TMS1 encodes a Hsp40-homologous protein with a DnaJ domain and an a_ERdj5_C domain found in protein disulfide isomerases (PDI). Purified TMS1 expressed in Escherichia coli (BL21 DE3) had the reductive activity of PDI. TMS1 was expressed in pollen grains, pollen tubes and other vegetative tissues, including leaves, stems and roots. Heat shock treatment at 37°C increased its expression levels in growing pollen tubes as well as in vegetative tissues. A knockout mutation in TMS1 grown at 30°C had greatly retarded pollen tube growth in the transmitting tract, resulting in a significant reduction in male fertility. Our study suggests that TMS1 is required for thermotolerance of pollen tubes in Arabidopsis, possibly by functioning as a co-molecular chaperone.