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Hsp100: AAA+ Family of Chaperones

Maintenance of protein quality control in the cell requires accomplishment of several steps that includes the folding of newly synthesized proteins, refolding and reactivation of unfolded and misfolded proteins, assembly and disassembly of macromolecular protein structures, and targeting abnormal and inactive proteins for degradation. Upon exposure of the cell to severe stress, many of the proteins lose their native conformation and get into non-specific interactions forming aggregates. Hsp100/Clp is a class of molecular chaperones that have the ability to solubilize almost any protein that becomes aggregated after severe stress. They are not required under normal growth conditions and are induced by extreme heat or other harsh stresses. ClpA was the first protein of Hsp100 class that was discovered as a component of ATP-dependent protease. It was named for its capacity to promote the proteolysis of casein (caseinolytic protease or Clp) (1).

Classification and Structure

Domain Organisation of Clp/Hsp100 protomers

Hsp100 class of chaperones belongs to the AAA+ superfamily of ATPases (6). The family is defined by the presence of a basic core of ~200–250 amino acids that comprise α-helical domain and a Walker-type nucleotide-binding domain. They have the ability to remodel the protein substrate in an ATP dependent manner (6). Hsp100 chaperones are categorized into two classes; class I proteins with two AAA+ modules and class II chaperones. Hsp104, bacterial ClpB and their distant relatives ClpA, ClpC belongs to class I whereas ClpX and HslU are a part of class II chaperones (3). Clp/Hsp100 proteins assemble into ring structures comprising six protomers. The crystal structure of ClpB shows the presence of an N-terminal domain (N domain) followed by a nucleotide binding domain (NBD-1), a middle domain (M domain) and a second nucleotide-binding domain (NBD-2). All class I Clp proteins share highly homologous NBDs (4). The N and M domains are distantly related when present. For example, ClpC has a shortened M domain, and ClpA lacks an M domain. On the other hand, in class II proteins, the single NBD is most homologous to NBD-2 of class I proteins. Three dimensional modeling of Hsp104/ClpB presents it as a hexamer with a three-tiered structure; one tier is formed by the N domain, one by NBD-1 and the third contains NBD-2 and the C domain (3). The cryo-electron microscopy maps of Hsp104 also show a large central cavity with an apical but not a distal pore. Additionally, Hsp104 contains a small 38 amino acid C-terminal domain (C domain) downstream of NBD-2 that is not present in ClpB. This region has been implicated in thermotolerance and hexamer assembly (3).

Hsp104 and ClpB: Machinery for protein disaggregation

Protein disaggregation by Clp/Hsp100 chaperone system

The nucleotide binding domains of Hsp104 and ClpB contain the characteristic Walker A and B motifs and sensor-1 and -2 motifs. Both the nucleotide binding domains are capable of binding and hydrolyzing ATP. ATP binding to the nucleotide binding domain stabilizes both the oligomeric state and interactions with the substrate (4). Hydrolysis of ATP provides energy for protein remodeling. The presence of M domain is unique for Hsp104 and ClpB proteins and distinguish it from other class I proteins. It consists of two anti-parallel coiled-coils resembling a two-bladed propeller and is inserted in the ATP binding domain. The presence of M domain is found to be essential for protein remodeling activity of the proteins (5).

Most of the disaggregation function by ClpB/Hsp104 is carried out in collaboration with the Hsp70/DnaK chaperone system, and both the systems act synergistically to remodel substrates that each can act on separately (4). The precise division of labour between Hsp70 and Hsp104 has not been established yet. It is possible that Hsp70 system functions early in the disaggregation process by assisting the extraction of polypeptides from aggregates and presenting unstructured regions of the aggregate to Hsp104. At the same time, it might modulate the ATPase cycle of Hsp104 to facilitate the protein remodeling process. The second possibility is the action of Hsp70 system late in the disaggregation process by assisting in reactivation of protein substrates that fail to spontaneously refold after the action of Hsp104. Although a direct demonstration of physical complexes between Hsp104 and Hsp70 has not been observed, hexameric form of ClpB is found to associate of DnaK through the helix-3 of its M domain (2).

Protein remodeling by Clp/Hsp100 chaperone system

Recent studies using yeast prion proteins have provided evidence that Hsp104 alone has the innate ability to remodel substrate proteins. High concentrations of Hsp104 block the assembly of yeast prion proteins into large insoluble fibers by eliminating the oligomeric intermediates that nucleate a fiber assembly, though the overall process requires interplay of other chaperone families (6). This observation is further substantiated by a study showing that both Hsp104/ClpB proteins have the intrinsic ability to unfold natively folded proteins independent of the Hsp70 system (7). However, in most situations, Hsp104 and ClpB work in collaboration with the Hsp70/DnaK chaperone system to remodel and disaggregate substrates ranging from specific ordered amyloid fibers such as prions to highly disordered insoluble protein aggregates. Further analysis is required to understand the interplay between Hsp104/ClpB and the Hsp70/DnaK system. Mutations in Hsp104/ClpB result in reduced thermotolerance, defects in cell division and accumulation of aggregated protein amyloids. Hsp104 potently inhibits Aβ42 amyloidogenesis, which is connected with Alzheimer’s disease. It also inhibits and reverses the formation of α-synuclein oligomers and fibers that are observed in Parkinson’s disease. Further Hsp104 antagonizes the degeneration of dopaminergic neurons induced by α-synuclein misfolding in the rat substantia nigra (3, 5). All these observations indicate towards development of Hsp104 as a therapeutic agent.

AAA+ proteases: ATP-powered protein degradation compartments

Protein degradation by Clp/Hsp100 chaperone system

They form an important subfamily of AAA+ machines that function in ATP-dependent protein degradation in cells ranging from bacteria to humans. ClpXP is a relatively simple and well-characterized AAA+ protease and is the basis for the understanding of other ATP-dependent proteases, including ClpAP, 42 ClpCP, HslUV, Lon, FtsH, PAN/20S, and the 26S proteasome. ClpAP was the first AAA+ protease that was identified. ClpAP consisted of ClpA that functioned as an ATPase and a separate peptidase ClpP. ClpP by itself, could digest small peptides but had no significant activity against proteins. Degradation of protein substrates required a combinatorial action of ClpA, ClpP, and ATP hydrolysis. ClpX was later identified as a AAA+ protease that carried out ATP-dependent proteolysis of substrates such as phage replication protein λO. Further, it was established that ClpXP and ClpAP differed in their substrate specificities and AAA+ modules of these proteases were responsible for recognizing substrates.

ClpXP is composed of two distinct proteins; a AAA+ ATPase called ClpX, and a peptidase called ClpP. ClpX recognizes unstructured peptide sequences in protein substrates and unfolds stable tertiary structure in the protein (7). The unfolded polypeptide chain is then spooled or translocated into a sequestered proteolytic compartment in ClpP for degradation into small peptide fragments (6). ClpX can also function as an ATP-dependent disassembly chaperone in the absence of ClpP. The biochemical functions of ClpX include binding substrates, adaptors and ClpP and finally protein unfolding and polypeptide translocation (3). Binding of ATP to ClpX results in productive interaction between ClpX and ClpP. However, unfolding and translocation of substrate requires both ATP binding and hydrolysis to power the changes in the enzyme conformation to drive these mechanical processes (4).

ClpXP recognizes its substrates by two mechanisms; first, is the direct recognition where ClpX recognizes protein substrates by binding to short unstructured peptide sequences, which are called degradation tags, degrons, or recognition signals. It can also bind and degrade proteins that contain the E. coli ssrA-tag sequence at their C terminus (5). Second mechanism is adaptor mediated recognition. Here two adaptor proteins, SspB and RssB work in concert with ClpXP to modulate proteolysis of specific substrates. SspB helps deliver ssrA- tagged proteins and additional substrates for ClpXP degradation (5). RssB is a two-component response regulator that controls degradation E. coli stationary-phase transcription factor σS. SspB specifically enhances ClpXP mediated degradation by lowering the KM and allows ClpXP to act even at low substrate concentration (5,6). In general, AAA+ class of proteases such as ClpX are involved in unfolding and degrading an enormous assortment of proteins with a wide range of structures and stabilities and thus function in maintaining protein quality control and numerous regulatory circuits in bacteria and eukaryotic organelles (1,5).


  1. Lund, P.A. (2001) Molecular chaperones in the cell. Oxford University Press, Oxford.
  2. Ben-Zvi AP, Goloubinoff P. (2001) Review: mechanisms of disaggregation and refolding of stable protein aggregates by molecular chaperones. J Struct Biol., 135(2), 84-93.
  3. Doyle, S.M. and Wickner, S. (2009) Hsp104 and ClpB: protein disaggregating machines. Trends Biochem Sci, 34, 40-48.
  4. Glover, J.R. and Lindquist, S. (1998) Hsp104, Hsp70, and Hsp40: a novel chaperone system that rescues previously aggregated proteins. Cell, 94, 73-82.
  5. Parsell, D.A. and Lindquist, S. (1993) The function of heat-shock proteins in stress tolerance: degradation and reactivation of damaged proteins. Annu Rev Genet, 27, 437-496.
  6. Bukau, B., Weissman, J. and Horwich, A. (2006) Molecular chaperones and protein quality control. Cell, 125, 443-451.
  7. Mayer, M.P. Gymnastics of molecular chaperones. Mol Cell, 39, 321-331.

Database Statistics

Total Number of Genomes
Numbers in Bacteria
Numbers in Archaea
Numbers in Protists
Numbers in Fungi
Numbers in Algae
Numbers in Plants
Numbers in Animals
Total Number of Hsp100 Proteins