ABSTRACT

Hsp70 chaperones provide an essential line of cellular defense against stress by assisting refolding of misfolded proteins, and they are furthermore required for a large variety of proteinfolding processes in unstressed cells (1, 2). This diversity in cellular roles, involving promiscuous but also selective substrate recognition (3), requires the activity of DnaJ cochaperones, which target Hsp70 proteins to their substrates (1, 4). DnaJ proteins constitute a large family of multidomain proteins, which share the signature J-domain required for cooperation with Hsp70 partner proteins. They differ with respect to other domains, which presumably permit association with specific Hsp70 target sites (4, 5). The existence of several DnaJ homologs in a cell, in conjunction with the specific substrates associated with them, is thought to be a major determinant for the functional diversity of Hsp70 proteins (1, 6).DnaJ proteins act by controlling the Hsp70 ATPase activity. Hsp70 alternates between the ATP state characterized by low affinity and fast exchange rates for substrates and the ADP state characterized by high affinity and low exchange rates (7). ATP hydrolysis is the rate-limiting step of the ATPase cycle that locks in substrates in a complex with Hsp70. This control of substrate binding by nucleotide requires communication between the N-terminal ATPase domain and the adjacent substrate-binding domain. The ATP hydrolysis step is the prime target for regulation of Hsp70 proteins, exerted by DnaJ proteins, which stimulate the hydrolysis rate by at least several hundred fold in the case of the Escherichia coli Hsp70, DnaK (1,6,(8)(9)(10)(11). A recent NMR study by using 15 N-labeled J-domain of DnaJ revealed that DnaJ interacts via its J-domain with the ATPase domain of DnaK (12)

No abstract

The 70 kDa heat shock proteins (the Hsp70 family) assist refolding of their substrates through ATP-controlled binding. We have analyzed mutants of DnaK, an Hsp70 homolog, altered in key residues of its substrate binding domain. Substrate binding occurs by a dynamic mechanism involving: a hydrophobic pocket for a single residue that is crucial for affinity, a two-layered closing device involving independent action of an alpha-helical lid and an arch, and a superimposed allosteric mechanism of ATP-controlled opening of the substrate binding cavity that operates largely through a beta-structured subdomain. Correlative evidence from mutational analysis suggests that the ADP and ATP states of DnaK differ in the frequency of the conformational changes in the alpha-helical lid and beta-domain that cause opening of the substrate binding cavity. The affinity for substrates, as defined by this mechanism, determines the efficiency of DnaJ-mediated and ATP hydrolysis mediated locking-in of substrates and chaperone activity of DnaK.

The DnaK chaperone system is involved in various cellular processes such as the control of the folded and oligomeric state of proteins under stress and non-stress conditions. In this study we functionally characterised the homologues of the DnaK system from Clostridium acetobutylicum DnaK, DnaJ, GrpE and OrfA were heterologously synthesised in Escherichia coli and affinity purified via a His-tag. By optimising the stoichiometry, we were able to refold guanidinium hydrochloride-denatured firefly luciferase in vitro with 22% of the yield obtained with the E. coli DnaK system. In addition, C. acetobutylicum DnaJ could stimulate the E. coli DnaK ATPase by a factor of 55. Furthermore, the DnaK system from C. acetobutylicum was able to prevent the aggregation of OrfA from C. acetobutylicum, which is similar to the repressor HrcA of CIRCE-regulated heat shock genes in Bacillus subtilis.