Characterization of a Lidless Form of the Molecular Chaperone DnaK

We examined the effect of deletion of different segments in the helical subdomain (the so-called "lid") of the DnaK peptide-binding domain on peptide binding and protein stability. At 25°C, wt DnaK and the deletion mutant proteins are able to stably bind peptides with similar affinity. However, at physiological (37°C) and stress (42°C) temperatures, removal of the N-terminal half of ␣B and the rest of the lid drastically decreases the ability of the protein to bind substrates. Differential scanning calorimetry and infrared spectroscopy show that this behavior is accompanied by destabilization of the peptide-binding domain. Our data suggest that the reversible interaction between the lid and ␤-sandwich subdomains of DnaK peptide-binding domain is required for the stabilization of the loops that form the peptide-binding site, which in turn modulates the protein affinity for peptide substrates. This interaction might have functional implications because it could prevent rebinding of the peptide substrate, which would be forced to fold.

Section: Resultsmentioning

confidence: 50%

mentioning

confidence: 99%

The Lid Subdomain of DnaK Is Required for the Stabilization of the Substrate-binding Site

Moro¹,

Fernández‐Sáiz²,

Muga

2004

“…DnaK or DnaK-ATP can bind with a low but not insignificant affinity to short hydrophobic peptides. ATP hydrolysis strongly increases the binding energy (4,5,7), and consequently, peptide release from DnaK-ADP may be more than a thousand times slower than from DnaK-ATP (19). Because the binding energy is proportional to the logarithm of the binding time, the affinity of DnaK-ADP for the polypeptide must be larger than that of DnaK-ATP by roughly 7 k B T. Highly stable native domains may require up to 20 k B T for their unfolding, but randomly misfolded domains are expected to be less specifically structured and therefore less stable than native domains.…”

Section: Passive and Active Molecular Mechanisms Of The Hsp70mentioning

confidence: 99%

Active Solubilization and Refolding of Stable Protein Aggregates By Cooperative Unfolding Action of Individual Hsp70 Chaperones

Ben‐Zvi

Rios

Dietler

et al. 2004

100

113

Hsp70 is a central molecular chaperone that passively prevents protein aggregation and uses the energy of ATP hydrolysis to solubilize, translocate, and mediate the proper refolding of proteins in the cell. Yet, the molecular mechanism by which the active Hsp70 chaperone functions are achieved remains unclear. Here, we show that the bacterial Hsp70 (DnaK) can actively unfold misfolded structures in aggregated polypeptides, leading to gradual disaggregation. We found that the specific unfolding and disaggregation activities of individual DnaK molecules were optimal for large aggregates but dramatically decreased for small aggregates. The active unfolding of the smallest aggregates, leading to proper global refolding, required the cooperative action of several DnaK molecules per misfolded polypeptide. This finding suggests that the unique ATP-fueled locking/unlocking mechanism of the Hsp70 chaperones can recruit random chaperone motions to locally unfold misfolded structures and gradually disentangle stable aggregates into refoldable proteins.

“…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.…”

mentioning

confidence: 99%

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

Horne

Genevaux³

et al. 2010

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 ...