Lyra Chang

Solution conformation of wild-type E. coli Hsp70 (DnaK) chaperone complexed with ADP and substrate.

DnaK is the canonical Hsp70 molecular chaperone protein from Escherichia coli. Like other Hsp70s, DnaK comprises two main domains: a 44-kDa N-terminal nucleotide-binding domain (NBD) that contains ATPase activity, and a 25-kDa substrate-binding domain (SBD) that harbors the substrate-binding site. Here, we report an experimental structure for wild-type, full-length DnaK, complexed with the peptide NRLLLTG and with ADP. It was obtained in aqueous solution by using NMR residual dipolar coupling and spin labeling methods and is based on available crystal structures for the isolated NBD and SBD. By using dynamics methods, we determine that the NBD and SBD are loosely linked and can move in cones of ±35° with respect to each other. The linker region between the domains is a dynamic random coil. Nevertheless, an average structure can be defined. This structure places the SBD in close proximity of subdomain IA of the NBD and suggests that the SBD collides with the NBD at this area to establish allosteric communication.

Heat Shock Protein 70 (Hsp70) as an Emerging Drug Target

Heat shock protein 70 (Hsp70) is a molecular chaperone that is expressed in response to stress. In this role, Hsp70 binds to its protein substrates and stabilize them against denaturation or aggregation until conditions improve.1 In addition to its functions during a stress response, Hsp70 has multiple responsibilities during normal growth; it assists in the folding of newly synthesized proteins,2, 3 the subcellular transport of proteins and vesicles,4 the formation and dissociation of complexes,5 and the degradation of unwanted proteins.6, 7 Thus, this chaperone broadly shapes protein homeostasis by controlling protein quality control and turnover during both normal and stress conditions.8 Consistent with these diverse activities, genetic and biochemical studies have implicated it in a range of diseases, including cancer, neurodegeneration, allograft rejection and infection. This review provides a brief review of Hsp70 structure and function and then explores some of the emerging opportunities (and challenges) for drug discovery. Hsp70 is Highly Conserved Members of the Hsp70 family are ubiquitously expressed and highly conserved; for example, the major Hsp70 from Escherichia coli, termed DnaK, is approximately 50% identical to human Hsp70s.9 Eukaryotes often express multiple Hsp70 family members with major isoforms found in all the cellular compartments: Hsp72 (HSPA1A) and heat shock cognate 70 (Hsc70/HSPA8) in the cytosol and nucleus, BiP (Grp78/HSPA5) in the endoplasmic reticulum and mtHsp70 (Grp75/mortalin/HSPA9) in mitochondria. Some of the functions of the cytosolic isoforms, Hsc70 and Hsp72, are thought to be redundant, but the transcription of Hsp72 is highly responsive to stress and Hsc70 is constitutively expressed. In the ER and mitochondria, the Hsp70 family members are thought to fulfill specific functions and have unique substrates, with BiP playing key roles in the folding and quality control of ER proteins and mtHsp70 being involved in the import and export of proteins from the mitochondria. For the purposes of this review, we will often use Hsp70 as a generic term to encompass the shared properties of the family members.

Chemical Manipulation of Hsp70 ATPase Activity Regulates Tau Stability

Alzheimers disease and other tauopathies have recently been clustered with a group of nervous system disorders termed protein misfolding diseases. The common element established between these disorders is their requirement for processing by the chaperone complex. It is now clear that the individual components of the chaperone system, such as Hsp70 and Hsp90, exist in an intricate signaling network that exerts pleiotropic effects on a host of substrates. Therefore, we have endeavored to identify new compounds that can specifically regulate individual components of the chaperone family. Here, we hypothesized that chemical manipulation of Hsp70 ATPase activity, a target that has not previously been pursued, could illuminate a new pathway toward chaperone-based therapies. Using a newly developed high-throughput screening system, we identified inhibitors and activators of Hsp70 enzymatic activity. Inhibitors led to rapid proteasome-dependent tau degradation in a cell-based model. Conversely, Hsp70 activators preserved tau levels in the same system. Hsp70 inhibition did not result in general protein degradation, nor did it induce a heat shock response. We also found that inhibiting Hsp70 ATPase activity after increasing its expression levels facilitated tau degradation at lower doses, suggesting that we can combine genetic and pharmacologic manipulation of Hsp70 to control the fate of bound substrates. Disease relevance of this strategy was further established when tau levels were rapidly and substantially reduced in brain tissue from tau transgenic mice. These findings reveal an entirely novel path toward therapeutic intervention of tauopathies by inhibition of the previously untargeted ATPase activity of Hsp70.

Binding of a Small Molecule at a Protein–Protein Interface Regulates the Chaperone Activity of Hsp70–Hsp40

Heat shock protein 70 (Hsp70) is a highly conserved molecular chaperone that plays multiple roles in protein homeostasis. In these various tasks, the activity of Hsp70 is shaped by interactions with co-chaperones, such as Hsp40. The Hsp40 family of co-chaperones binds to Hsp70 through a conserved J-domain, and these factors stimulate ATPase and protein-folding activity. Using chemical screens, we identified a compound, 115-7c, which acts as an artificial co-chaperone for Hsp70. Specifically, the activities of 115-7c mirrored those of a Hsp40; the compound stimulated the ATPase and protein-folding activities of a prokaryotic Hsp70 (DnaK) and partially compensated for a Hsp40 loss-of-function mutation in yeast. Consistent with these observations, NMR and mutagenesis studies indicate that the binding site for 115-7c is adjacent to a region on DnaK that is required for J-domain-mediated stimulation. Interestingly, we found that 115-7c and the Hsp40 do not compete for binding but act in concert. Using this information, we introduced additional steric bulk to 115-7c and converted it into an inhibitor. Thus, these chemical probes either promote or inhibit chaperone functions by regulating Hsp70-Hsp40 complex assembly at a native protein-protein interface. This unexpected mechanism may provide new avenues for exploring how chaperones and co-chaperones cooperate to shape protein homeostasis.

Facilitating Akt Clearance via Manipulation of Hsp70 Activity and Levels

Members of the 70-kDa heat shock family can control and manipulate a host of oncogenic client proteins. This role of Hsp70 in both the folding and degradation of these client proteins makes it a potential drug target for certain forms of cancer. The phenothiazine family of compounds, as well as the flavonoid myricetin, was recently shown to inhibit Hsp70-ATPase activity, whereas members of the dihydropyrimidine family stimulated ATPase function. Akt, a major survival kinase, was found to be under the regulation of Hsp70, and when the ATPase activity of Hsp70 was increased or decreased by these compounds, Akt levels were also increased or decreased. Also, increasing Hsp70 levels concurrent with inhibition of its ATPase function synergistically reduced Akt levels to a greater extent than either manipulation alone, providing new insights about client fate decisions. Akt reductions mediated by Hsp70 inhibitors were prevented when Hsp70 expression was silenced with small interfering RNA. Inhibiting Hsp70 ATPase function produced cytotoxic events only in breast cancer cell lines where Akt dysfunction was previously shown, suggesting therapeutic specificity depending on the Hsp70 client profile. Thus, increasing Hsp70 levels combined with inhibiting its ATPase function may serve to dramatically reduce Akt levels and facilitate cell death in certain types of cancer.

Mutagenesis Reveals the Complex Relationships between ATPase Rate and the Chaperone Activities of Escherichia coli Heat Shock Protein 70 (Hsp70/DnaK)

The Escherichia coli 70-kDa heat shock protein, DnaK, is a molecular chaperone that engages in a variety of cellular activities, including the folding of proteins. During this process, DnaK binds its substrates in coordination with a catalytic ATPase cycle. Both the ATPase and protein folding activities of DnaK are stimulated by its co-chaperones, DnaJ and GrpE. However, it is not yet clear how changes in the stimulated ATPase rate of DnaK impact the folding process. In this study, we performed mutagenesis throughout the nucleotide-binding domain of DnaK to generate a collection of mutants in which the stimulated ATPase rates varied from 0.7 to 13.6 pmol/μg/min−1. We found that this range was largely established by differences in the ability of the mutants to be stimulated by one or both of the co-chaperones. Next, we explored how changes in ATPase rate might impact refolding of denatured luciferase in vitro and found that the two activities were poorly correlated. Unexpectedly, we found several mutants that refold luciferase normally in the absence of significant ATP turnover, presumably by increasing the flexibility of DnaK. Finally, we tested whether DnaK mutants could complement growth of ΔdnaK E. coli cells under heat shock and found that the ability to refold luciferase was more predictive of in vivo activity than ATPase rate. This study provides insights into how flexibility and co-chaperone interactions affect DnaK-mediated ATP turnover and protein folding.

Bicinchoninic acid (BCA) assay in low volume.

The BCA assay is a colorimetric method for estimating protein concentration. In 96-well plates, the relationship between protein content and absorbance is nearly linear over a wide range; however, performance is reduced in lower volume. To overcome this limitation, we performed the BCA assays in opaque, white 384-well plates. These plates emit fluorescence between 450-600 nm when excited at 430 nm; thus, their fluorescence is quenched by the BCA chromophore (λ(max) 562 nm). This arrangement allowed accurate determination of protein content using only 2 μL of sample. Moreover, soluble flourescein could replace the white plates, creating a homogenous format.

High-Throughput Screen for Escherichia coli Heat Shock Protein 70 (Hsp70/DnaK): ATPase Assay in Low Volume by Exploiting Energy Transfer

Members of the heat shock protein 70 (Hsp70) family of molecular chaperones are emerging as potential therapeutic targets. Their ATPase activity has classically been measured using colorimetric phosphate detection reagents, such as quinaldine red (QR). Although such assays are suitable for 96-well plate formats, they typically lose sensitivity when attempted in lower volume due to path length and meniscus effects. These limitations and Hsp70’s weak enzymatic activity have combined to create significant challenges in high-throughput screening. To overcome these difficulties, the authors have adopted an energy transfer strategy that was originally reported by Zuck et al. (Anal Biochem 2005;342:254-259). Briefly, white 384-well plates emit fluorescence when irradiated at 430 nm. In turn, this intrinsic fluorescence can be quenched by energy transfer with the QR-based chromophore. Using this more sensitive approach, the authors tested 55,400 compounds against DnaK, a prokaryotic member of the Hsp70 family. The assay performance was good (Z′ ~0.6, coefficient of variation ~8%), and at least one promising new inhibitor was identified. In secondary assays, this compound specifically blocked stimulation of DnaK by its co-chaperone, DnaJ. Thus, this simple and inexpensive adaptation of a colorimetric method might be suitable for screening against Hsp70 family members.

Isoform-selective Genetic Inhibition of Constitutive Cytosolic Hsp70 Activity Promotes Client Tau Degradation Using an Altered Co-chaperone Complement

Background: High levels of constitutive heat shock protein 70 (Hsc70) preserve Tau levels, possibly contributing to neuropathology. Results: A dominant negative Hsc70 (DN-Hsc70) variant mimics small-molecule inhibition and facilitates Tau clearance by altering the associated interactome. Conclusion: DN-Hsc70 recruits a pro-degradation chaperome complement to the Tau-Hsc70 complex. Significance: Selective Hsc70 inhibition could have a therapeutic benefit in tauopathies including Alzheimer disease. The constitutively expressed heat shock protein 70 kDa (Hsc70) is a major chaperone protein responsible for maintaining proteostasis, yet how its structure translates into functional decisions regarding client fate is still unclear. We previously showed that Hsc70 preserved aberrant Tau, but it remained unknown if selective inhibition of the activity of this Hsp70 isoform could facilitate Tau clearance. Using single point mutations in the nucleotide binding domain, we assessed the effect of several mutations on the functions of human Hsc70. Biochemical characterization revealed that one mutation abolished both Hsc70 ATPase and refolding activities. This variant resembled the ADP-bound conformer at all times yet remained able to interact with cofactors, nucleotides, and substrates appropriately, resembling a dominant negative Hsc70 (DN-Hsc70). We then assessed the effects of this DN-Hsc70 on its client Tau. DN-Hsc70 potently facilitated Tau clearance via the proteasome in cells and brain tissue, in contrast to wild type Hsc70 that stabilized Tau. Thus, DN-Hsc70 mimics the action of small molecule pan Hsp70 inhibitors with regard to Tau metabolism. This shift in Hsc70 function by a single point mutation was the result of a change in the chaperome associated with Hsc70 such that DN-Hsc70 associated more with Hsp90 and DnaJ proteins, whereas wild type Hsc70 was more associated with other Hsp70 isoforms. Thus, isoform-selective targeting of Hsc70 could be a viable therapeutic strategy for tauopathies and possibly lead to new insights in chaperone complex biology.

Identification of Key Hinge Residues Important for Nucleotide-Dependent Allostery in E. coli Hsp70/DnaK

DnaK is a molecular chaperone that has important roles in protein folding. The hydrolysis of ATP is essential to this activity, and the effects of nucleotides on the structure and function of DnaK have been extensively studied. However, the key residues that govern the conformational motions that define the apo, ATP-bound, and ADP-bound states are not entirely clear. Here, we used molecular dynamics simulations, mutagenesis, and enzymatic assays to explore the molecular basis of this process. Simulations of DnaKs nucleotide-binding domain (NBD) in the apo, ATP-bound, and ADP/Pi-bound states suggested that each state has a distinct conformation, consistent with available biochemical and structural information. The simulations further suggested that large shearing motions between subdomains I-A and II-A dominated the conversion between these conformations. We found that several evolutionally conserved residues, especially G228 and G229, appeared to function as a hinge for these motions, because they predominantly populated two distinct states depending on whether ATP or ADP/Pi was bound. Consistent with the importance of these “hinge” residues, alanine point mutations caused DnaK to have reduced chaperone activities in vitro and in vivo. Together, these results clarify how sub-domain motions communicate allostery in DnaK.