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Hsp40: An Obligate Co-chaperone

Hsp40 family of heat shock proteins are the critical drivers of Hsp70 function. These 40 kD proteins are ubiquitously expressed in all organisms ranging from bacteria to higher eukaryotes. The family of Hsp40 is quite diverse and range from 116 amino acids DnaJC19 to 2243 amino acids DnaJC13. Depending on the physiological complexity of the organisms, the presence of Hsp40s varies from 22 in yeast to 44 in humans. Clearly, the number of Hsp40s in the cell far exceeds that of Hsp70s. For example, a mammalian cell has only 11 Hsp70s but 41 Hsp40s (1). Additionally, Hsp40s also show a much higher sequence and structural divergence than Hsp70 in the cell. This is consistent with the idea that Hsp40 family has evolved to aid in the versatility and multi-functionality of the Hsp70 chaperone system.

Domain organization

domain
Domain organisation of Hsp40/J-protein

All Hsp40s are characterized by the presence of a canonical J-domain and are hence also termed as the J-proteins. J-domain is approximately 70 amino acids α-helical signature region that derives its nomenclature after the E. Coli Hsp70 co-chaperone DnaJ (2). The structure of J-domain is conserved across all kingdoms. Even highly diverged J-proteins across species retain remarkable three dimensional structural fold similarity at the J-domain region. It consists of four helices with tightly packed helix II and III in antiparallel orientation. Both these helices are connected via a flexible loop containing a highly conserved and functionally critical HPD signature motif that is characteristic of J-domain (3,4). The HPD tripeptide is essential for stimulation of ATPase activity of Hsp70 (4). Mutations altering either the histidine or whole tripeptide abolishes the stimulatory activity of the J-domain. However, additional residues within helices II and III and in the intervening loops are required for the in vivo function of J-protein (4). This J-domain is remarkably similar in most of the J-proteins even they are highly diverged across species. Other than the J-domain Hsp40s might contain ‘a G/F region’, ‘a Zinc-finger domain’ and a variable ‘C-terminal domain’. G/F region is glycine/phenylalanine rich linker that separates the J-domain. It is required for stability and precise positioning of the J-domain during its interaction with Hsp70 (1,2). The zinc-finger domain contains four CXXCXGXG repeats present in two separate clusters where each cluster co-ordinates with a zinc ion. This domain is crucial in sequestering the denatured substrate and assisting Hsp70 during the protein folding reaction (4). The C-terminal region is less conserved and consists of a β-sandwich surrounded by a short C-terminal α-helix, followed by sequences essential for dimerization. This region plays a crucial role in substrate binding and its sequestration into Hsp70 during chaperone cycle and is thought to provide specificity for the Hsp70:J-protein machine (5).


J-protein classification

domain
Structural classification of J-proteins

Based on the domain organization, J-proteins are classified into 4 types namely; Type I, Type II, Type III and Type IV. Type I J-proteins show the presence of all domains found in DnaJ (2). They contain an N-terminal J-domain that is separated from the rest of the protein by a 50-100 amino acids long flexible linker ‘G/F region’. Distal to G/F region is the zinc-binding cysteine-rich sequence named as ‘Zinc-finger domain’ which is the signature motif of type I proteins and distinguishes it from other types of J-proteins. Zinc-finger domain is followed by the C-terminal domain (1,2). Type II proteins possess all the domains except the zinc-finger domain. In contrast, Type III J-proteins contain a C-terminal J-domain and lack both G/F and zinc-finger domains. Type IV constitutes a group of recently identified proteins that are conspicuous by the absence of HPD motif in their primary sequence (2). Instead, they have a fewer conserved DKE motif conserved DKE motif and is also termed as J-like proteins. The Type IV proteins similar to their Type III counterparts have their J-like domain towards C-terminus of the protein (2).

Mode of Action

40cycle
Model of Hsp70:J-protein chaperone cycle

During protein folding process Hsp70s do not function individually. They form a complex with J-proteins to form the folding-machine (5). Since the intrinsic ATPase activity of Hsp70 is very weak for stoichiometric coupling to the substrate binding-release cycle, J-proteins act as accessory factors for Hsp70 and can serve more than one Hsp70 molecule (3,6). They stimulate Hsp70 ATPase activity by transiently interacting with it (3,4). In a chaperone cycle, Hsp70 proteins have two distinct conformations; ATP-bound state that transiently interacts with client proteins and ADP-state that stably bind to the substrate (6). A client protein interacts with Hsp70 in its ATP bound state. Few subclasses of J-proteins directly sequester substrates into the peptide-binding cleft of an Hsp70 and is coupled to stimulation of ATP hydrolysis by J-domains of Hsp40s that interact with the ATPase domain of Hsp70 (3,5). The conversion of Hsp70 to ADP bound state stabilizes the interaction of Hsp70 with the client protein. Nucleotide exchange factors replace ADP with ATP resulting in dissociation of the bound peptide and hence prime Hsp70 for a second cycle of interaction (3,5). The repeated cycles of interaction with the substrate ensure its proper folding and prevent it from aggregating (5). Therefore, J-proteins play a critical role in regulating the chaperone cycle catalytically and thereby control many physiological functions in the cell.


References

  1. Kampinga, H.H. and Craig, E.A. The HSP70 chaperone machinery: J proteins as drivers of functional specificity. Nat Rev Mol Cell Biol, 11, 579-592.
  2. Rajan, V.B. and D'Silva, P. (2009) Arabidopsis thaliana J-class heat shock proteins: cellular stress sensors. Funct Integr Genomics, 9, 433-446.
  3. Laufen, T., Mayer, M.P., Beisel, C., Klostermeier, D., Mogk, A., Reinstein, J. and Bukau, B. (1999) Mechanism of regulation of hsp70 chaperones by DnaJ cochaperones. Proc Natl Acad Sci U S A, 96, 5452-5457.
  4. Walsh, P., Bursac, D., Law, Y.C., Cyr, D. and Lithgow, T. (2004) The J-protein family: modulating protein assembly, disassembly and translocation. EMBO Rep, 5, 567-571.
  5. Craig, E.A., Huang, P., Aron, R. and Andrew, A. (2006) The diverse roles of J-proteins, the obligate Hsp70 co-chaperone. Rev Physiol Biochem Pharmacol, 156, 1-21.
  6. Misselwitz, B., Staeck, O. and Rapoport, T.A. (1998) J proteins catalytically activate Hsp70 molecules to trap a wide range of peptide sequences. Mol Cell, 2, 593-603.

Database Statistics

Total Number of Genomes
277
Numbers in Bacteria
508
Numbers in Archaea
47
Numbers in Protists
406
Numbers in Fungi
662
Numbers in Algae
242
Numbers in Plants
854
Numbers in Animals
1182
Total Number of Hsp40 Proteins
3901