A disulfide-bonded DnaK dimer is maintained in an ATP-bound state

Abstract

DnaK, a major Hsp70 molecular chaperones in Escherichia coli, is a widely used model for studying Hsp70s. We recently solved a crystal structure of DnaK in complex with ATP and showed that DnaK was packed as a dimer in the crystal structure. Our previous biochemical studies supported the formation of a specific DnaK dimer as observed in the crystal structure in solution in the presence of ATP and suggested an important role of this dimer in efficient interaction with Hsp40 co-chaperones. In this study, we dissected the biochemical properties of this DnaK dimer. To restrict DnaK in this dimer form, we mutated two residues on the dimer interface to cysteine, A303C, and H541C. Upon oxidation, this DnaK-A303C-H541C protein formed a specific dimer linked by disulfide bonds formed between A303C and H541C only in the presence of ATP, consistent with the crystal structure. Intriguingly, this disulfide-bond-linked dimer of DnaK-A303C-H541C has reduced ATPase activity and decreased affinity for peptide substrate. More interestingly, unlike wild-type DnaK, the peptide substrate-binding kinetics of this dimer is drastically accelerated even in the absence of ATP, suggesting this dimer is restricted in an ATP-bound conformation regardless of nucleotide bound, which was further supported by our analysis using tryptophan fluorescence and ATP-induced peptide release. Thus, formation of the dimer restricted DnaK in an ATP-bound state and blocked the progression through the chaperone cycle. Productive progression through the chaperone cycle requires the dissociation of this transient dimer. Surprisingly, a significantly compromised interaction with Hsp40 co-chaperone was observed for this disulfide-bond-linked dimer. Thus, dissociation of this DnaK dimer is equally crucial for efficient Hsp40 interaction. An initial interaction between Hsp70 and Hsp40 requires the formation of DnaK dimer; but a stable Hsp70-Hsp40 interaction may follow the dissociation of the dimer.

Human Stress-inducible Hsp70 Has a High Propensity to Form ATP-dependent Antiparallel Dimers That Are Differentially Regulated by Cochaperone Binding*

“…Thus, transient stabilization of the SBD␣ subdomain of an Hsp70 protomer by its docking onto the NBD domain of the partner Hsp70 molecule might provide a time window for productive Hsp40-mediated substrate positioning into SBD␤ before the SBD␣ subdomain closes over SBD␤. This hypothesis is consistent with the paradoxical observation that the DnaJ-DnaK interaction relies on both ATP-dependent DnaK dimerization (25) and dimer dissociation allowing free movement of the SBD␣ subdomain (26). Additionally, our SEC analysis of Hsp70 ATP-dependent dimers coincubated with Hsp40 ( Fig.…”

“…Conversely, the inhibitory role of Tomm34 on Hsp70-mediated refolding activity (29) can be attributed to its ability to dissociate Hsp70 ATP-bound dimers ( Fig. 7B and 7C) necessary for substrate capture during effective Hsp70-Hsp40 interaction (18,25,26,82,83). We have previously suggested that the additional Tomm34-Hsp70 binding site is located between residues 533-543 in the SBD␣ subdomain (29).…”

“…Formation and disassembly of a small population of ATPdependent DnaK dimers plays a critical role in DnaJ-regulated DnaK ATPase activity (25,26). Moreover, conservancy of residues involved in mediating homo-dimerization of ATPbound DnaK in its eukaryotic homologs supports a functional role of the ATP-dependent dimer (52).…”

“…Therefore, we speculate that Hsp40-mediated substrate transfer to ATPbound Hsp70 requires chaperone transient dimerization. Evidence exists suggesting that substrate binding to SBD␤ needs to be followed by SBD␣ closing over SBD␤ for effective Hsp40/substrate-stimulated ATPase activity of Hsp70 (11,26). Thus, transient stabilization of the SBD␣ subdomain of an Hsp70 protomer by its docking onto the NBD domain of the partner Hsp70 molecule might provide a time window for productive Hsp40-mediated substrate positioning into SBD␤ before the SBD␣ subdomain closes over SBD␤.…”

“…Considering the recently discovered regulatory role of ATPinduced dimerization for Hsp70 chaperoning activity (Hsp40 binding) (25,26) and multichaperone complex formation (27), differential effects of TPR cochaperone binding to Hsp70 ATP-bound dimers (Fig. 7) are likely to be important for overall activity.…”