HSP40: History

Heat shock proteins (HSPs) were first discovered in the early 1960s in experiments on the fruitfly Drosophila melanogaster where dramatic changes in the puffering pattern of the polytene chromosomes in Drosophila’s salivary glands could be observed upon exposure to heat shock 19,20,21. Nearly a decade later, Alfred Tissières observed an upregulated expression of a set of mRNAs and polypeptides in Drosophila after exposure to heat shock 22. The universality of this response from bacteria to humans became apparent shortly thereafter 23. Expression of HSPs was found as being induced after exposure to environmental stressors and could be demonstrated subsequently in any cellular organism allowing cells to survive different lethal conditions 24. Environmental stressors comprise physical as well as chemical and biological insults such as hypoxia, heavy metals, pH shift, nutrient deprivation and irradiation as well as organics, heavy metals and oxidants, infections, inflammation and diseases 24,25,26,27,28,29. This HSP induction is referred to as the heat shock response (HSR) that is ubiquitous across the bacterial, archaeal and eukaryotic kingdoms 30, 31. The painstaking analysis of the limited number of proteins firstly identified by heat shock induction in D. melanogaster and in Escherichia coli led to the finding that DnaJ is also a member of the heat shock class of proteins.

Members of the 40 kDa heat shock protein (DNAJ/HSP40) family are characterized by the presence of the remarkably conserved J-domain. This J-domain was first identified in E. coli DnaJ and bears a conserved HPD tripeptide as the signature motif 32. Historically, HSP40s/DNAJs have been classified into three subtypes (1, 2 and 3; also referred as to subfamily A, B, and C) based on differences in the conserved regions found in their amino acid sequences 33,34,35. DnaJ has originally been reported to stimulate the ATPase activity of the bacterial Hsp70 homolog DnaK and to initiate bacteriophage λ DNA replication 32, 36. Since its initial characterization, DnaJ has been shown to interact not only with DnaK but also with the nuclear exchange factor GrpE (GroP-like gene E) in order to facilitate λ phage 37, 38, plasmid P1 39, 40 and DNA replication 41, folding of nascent polypeptides 42, protein export 43 and repair 44, 45 as well as the assembly of macromolecular complexes implicated in flagellum synthesis in E. coli 46. Of note, DnaJ also possesses an autonomous chaperone activity which has been demonstrated by the group of Martin Wiedmann suggesting DnaJ-mediated binding of polypeptides prior to DnaK recruitment 42. The zinc finger (ZF) domains within DnaJ and its yeast ortholog Ydj1 have been shown to represent the structural requirements for this “holdase” activity enabling the interaction of DnaJ and Ydj1 with unfolded polypeptides thereby preventing their aggregation 47, 48. Ohtsuka et al. discovered a heat-inducible 40 kDa heat shock protein, termed hsp40, in mammalian and avian cells that was also found to be induced by transient metals and azetidine carboxylic acid 49.

The nuclear magnetic resonance (NMR) structures of DnaJ and its human ortholog DnaJB1/Hdj1 have been solved in the mid-1990s 50,51,52,53. The putative interaction site between the DnaJ J-domain and DnaK could be identified by Suh and co-workers in 1998 54. Heteronuclear NMR experiments then recognized critical amino acid residues located in helix II and the conserved HPD motif of the J-domain as interaction partners for the ATPase domain of DnaK 55. These findings were supported by the observation that a synthetic peptide bearing helix II and the HPD motif is able to block the interaction between Ydj1 and the Hsp70 chaperone Ssa1 in yeast 4. The crystal structure of E. coli HscB/Hsc20 was solved shortly thereafter 56. In 2004, Gruschus et al. constructed a model structure of the complex composed of DnaJC6/auxilin and Hsc70 leading to the identification of critical amino acids involved in forming H bonds and salt bridges between the HPD motif in DnaJC6/auxilin and the ATPase domain of Hsc70 57. Borges and co-workers determined the quaternary structure of human DnaJA1/Hdj2 and DnaJB4/Hlj1 in a low resolution structural study revealing the dimeric nature of both molecules 58. While DnaJA1/Hdj2 was shown to form a compact dimer with the N- and C-termini of the monomers facing each other, DnaJB4/Hlj1 formed a dimer in which only the corresponding C-termini interacted. In order to provide insight into the interaction of DNAJs/HSP40s with protein substrates, the group of Fernando Moro characterized the structure of the complex between DnaJ and its client protein RepE, the replication initiation factor of plasmid mini-F 59. This was the first described structure of a complex between a member of the DNAJ/HSP40 family and a client protein. In this study, DnaJ has been found to induce conformational changes in dimeric RepE thereby enhancing its affinity for DNA 60. The model also revealed an intrinsic plasticity of the DnaJ dimer enabling DnaJ to adapt to distinct protein substrates 60. The crystal structure of human DnaJC3/p58IPK has been determined recently by Svärd et al. demonstrating that DnaJC3/p58IPK bears nine N-terminal tetratricopeptide repeat (TPR) motifs in addition to the conserved J-domain attached to the very end of the TPR domain via a flexible linker 61. From the structure the putative binding site for HspA5 (Grp78, BiP, Kar2) based on the position of the conserved HPD motif could be determined.

The first crystal structure of a complete functional dimeric DnaJ from Thermus thermophilus was determined recently and the mobility of its individual domains in solution was investigated by Barends and collaborators 62.