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.allostery ͉ dipolar couplings ͉ dynamics ͉ NMR ͉ structure H sp70 (heat shock 70 kDa) chaperone proteins are central to protein folding, refolding, and trafficking in organisms ranging from Archae to Homo sapiens, both at normal and at stressed conditions (for a review, see ref. 1). Recently, Hsp70s have been linked to breast and colon cancer (2) and to diseases such as Alzheimer's (3), Parkinson's (4), and Huntington's (5) diseases. In this report, DnaK, the canonical Hsp70 molecular chaperone protein from Escherichia coli, is studied. In the ADP state, DnaK, like other Hsp70s, binds to exposed hydrophobic residues of unfolded or partially misfolded proteins. Upon ATP binding, which induces an allosteric conformational change, DnaK releases the client protein (6). This process is tightly regulated by cochaperone proteins (7). DnaK consists of three subdomains. The structure of the nucleotide-binding domain (NBD, residues 1-370), was solved by crystallography (8). It competitively binds ATP and ADP and can slowly hydrolyze ATP (9). Structures for the 15-kDa substratebinding domain (SBD, residues 390-600) were solved in different forms by crystallography (10) and NMR (11-13). It harbors the hydrophobic substrate-binding cleft. Here, this subdomain is referred to as BETA. A subsequent 10-kDa subdomain of ␣-helical structure (residues 510-638) was characterized by NMR (14) and crystallography (15). This subdomain, referred to as the LID, plays a key role in regulating the kinetics of substrate binding (16,17).Recently, structures have become available comprising both the NBD and SBD. Our group has used NMR methods to determine the global 3D solution structure of an NBD-SBD construct (residues 1-501) of Thermus thermophilus DnaK (18). A crystal structure of Bos taurus Hsc70 (residues 1-554 and E213A/D214A) was reported (19). A crystal structure for Geobacillus kaustophilus DnaK (residues 1-509) was determined (20). Furthermore, a crystal structure of Saccharomyces cerevisiae Hsp110 (2-659), which is a Hsp70 ho...

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 ConservedMembers 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. Domain Architecture and Substrate Binding of Hsp70All members of the Hsp70 family have an N-terminal nucleotide binding domain (NBD) (~40 kDa) and a C-terminal substrate-binding domain (SBD) (~25 kDa) connected by a short linker ( Figure 1A).10 The NBD consists of two subdomains, I and II, which are further divided into regions a and b. The Ia and IIa subdomains interact with ATP through a nucleotide-binding cassette related to those of hexokinase, actin and glycerol kinase.11 , 12 The SBD consists of a 10-kDa α-helix subdomain and a 15-kDa β-sandwich. Crystal structures suggest that substrate peptides are bound in an extended conformation between * Correspondence can be addressed to: Jason E. Gestwicki, University of Michigan, Life Sciences Institute, 210 Washtenaw Ave, Ann Arbor, MI 48109-2216, P (734) 615-9537, gestwick@umich.edu. loops of the β-sandwich and that the α-heli...

Alzheimer's 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.

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.Heat shock protein 70 (Hsp70) is a member of a ubiquitously expressed family of molecular chaperones that are involved in protein homeostasis. In its role as a mediator of protein fate, this chaperone has been linked to multiple tasks, including roles in de novo protein folding, subcellular trafficking, protein disaggregation, proteasome-mediated degradation, and autophagy (1-6). In addition, Hsp70 has been linked to numerous diseases, especially cancer and disorders of protein folding (7). Thus, there is interest in better understanding the biology of Hsp70 in order to test its potential as a therapeutic target (8).To accomplish its various chaperone functions, Hsp70 physically interacts with the exposed hydrophobic residues of polypeptides via its C-terminal substrate-binding domain (SBD). NIH-PA Author ManuscriptHydrolysis of ATP in the adjacent, N-terminal nucleotide-binding domain (NBD) propagates an allosteric change to the SBD, resulting in an approximately 10-fold enhancement in substrate affinity (9-12). These findings suggest an important role for the nucleotide state in controlling the interactions of Hsp70 with misfolded substrates. Consistent with the proposed importance of nucleotide turnover, a family of essential cochaperones, the Hsp40s, is known to tightly regulate the ATPase rate of Hsp70. These cochaperones are defined by the presence of a conserved, 60 amino acid J-domain. Interaction of the J-domain with the NBD of a Hsp70 stimulates its ATP hydrolysis and favors tight association with bound substrates. For example, the J-domain containing co-chaperone, DnaJ, stimulates the nucleotide hydrolysis rate o...

No abstract