The Hsp40 J-domain Stimulates Hsp70 When Tethered by the Client to the ATPase Domain

Abstract

Hsp70-family molecular chaperones have numerous constitutive and stress-induced roles that involve binding to short hydrophobic stretches within a client protein. Protein folding, protein translocation, and protein disaggregation are a few of the functions that Hsp70s carry out under normal conditions within the cell (1-4).The two major activities of Hsp70s are located in the two major domains of the protein. The N-terminal 44-kDa domain binds and hydrolyzes ATP, and the 23-kDa peptide binding domain binds client proteins (5). The peptide binding domain is divided into the ␤-sandwich subdomain where client binding occurs and the ␣-helical subdomain, which acts as a lid on the ␤-sandwich subdomain (6). The two major domains are connected by a highly conserved linker region.Escherichia coli Hsp70/DnaK is the most extensively studied member of the Hsp70s. The behavior of DnaK is like other Hsp70s in that its binding to client proteins is regulated by binding and hydrolysis of ATP. The binding of ATP converts DnaK to the low affinity conformation. This form of the protein binds and releases client proteins quickly, on the time scale of seconds (7,8). Hydrolysis of ATP slows the off-rate of client proteins by 3 orders of magnitude compared with when ATP is bound (9). Previous studies have shown that a portion of the lid subdomain of DnaK modulates the allosteric behavior. For example, for a lidless form of DnaK (DnaK(1-517)) containing only a portion of helix A, the ATP-stimulated release of peptide is ϳ40-fold faster than for wild-type DnaK (10).The DnaK client binding cycle is regulated by a nucleotide exchange factor, GrpE (11). GrpE interacts with the ATPase domain of DnaK to catalyze the release of ADP (12). Upon release of ADP, DnaK binds ATP, which stimulates the release of bound client.The structurally and functionally distinct E. coli Hsp40/DnaJ has been described as a co-chaperone for Hsp70/DnaK, and the two proteins are said to operate as a single chaperone machine (13). DnaJ binds to client proteins, and it stimulates the DnaK ATPase activity. The two functions of DnaJ have been assigned to the C terminus and N terminus of the molecular chaperone, respectively. The N-terminal 75-residue J-domain (Jd) 2 is responsible for stimulation of DnaK ATPase activity (14, 15). The Jd is also able to couple ATP hydrolysis to peptide capture in DnaK (15). Two distinct models have been suggested for how the Jd couples the DnaK ATPase and peptide binding activities, client protein-recruitment, and triggered capture. In the recruitment model, the Jd brings an associated client protein to . In the triggered-capture model, the client protein brings the Jd to DnaK (14,19,20). After formation of the three-component complex, the Jd stimulates ATP hydrolysis by DnaK, which firmly stabilizes the client-bound conformation of DnaK. The triggered-capture model more accurately described the role of the Jd in binding of a model client, the peptide p5, because the association rate of the p5 with DnaK was only modestly increased by ...

Heat shock protein 70 kDa chaperone/DnaJ cochaperone complex employs an unusual dynamic interface

“…There is a small, but very significant, difference between the data of Landry and coworkers (13,23) and ours: They also observed chemical shift changes for residue Asp35 of the conserved HPD loop. However, we found that shifts for the NH signal of Asp35 can also be caused by very small changes in ionic strength.…”

Allostery in the Hsp70 Chaperone Proteins

“…This J-fusion bound much tighter to DnaK in the presence of ATP (0.2 nM) than in the absence of nucleotide (30 nM). A later paper of the same group using the same construct and using NMR spectroscopy to monitor binding to several DnaK constructs in different nucleotide states confirmed their earlier observations 159 . SPR experiments by Gross and co-workers 155 strongly suggest that wt-DnaJ binds tightly to the ATP state (K D = 70 nM) but does not bind to DnaK in the ADP state (i.e.…”

Chaperones and chaperone–substrate complexes: Dynamic playgrounds for NMR spectroscopists

“…Based on interaction studies using 15 N-line broadening analysis of DnaK with a chimeric protein consisting of a J-domain and a DnaK client protein (p5), it was shown that the chimera showed a stronger binding affinity towards DnaK and revealed novel insight into the functional interplay of DnaJ and DnaK. In conclusion, these results suggest that the J-domain shifts the equilibrium of DnaK to a substrate-bound form, enabling more productive binding of the substrate with subsequent stimulation of the ATPase function of DnaK [161]. Subsequent determination of the solution structure of the complex of DnaK with the J-domain of DnaJ in the ADP state and in the presence of a substrate peptide confirmed the interaction via helix II of the J-domain with a negatively charged loop in the Hsp70 nucleotide-binding domain.…”