Bacterial J-Domains with C-Terminal Tags Contact the Substrate Binding Domain of DnaK and Sequester Chaperone Activity

Functional interactions between the molecular chaperone DnaK and cofactor J-proteins (DnaJs), as well as their homologs, are crucial to the maintenance of proteostasis across cell types. In the bacterial pathogen Mycobacterium tuberculosis, DnaK–DnaJ interactions are essential for cell growth and represent potential targets for antibiotic or adjuvant development. While the N-terminal J-domains of J-proteins are known to form important contacts with DnaK, C-terminal domains have varied roles. Here, we have studied the effect of adding C-terminal tags to N-terminal J-domain truncations of mycobacterial DnaJ1 and DnaJ2 to promote additional interactions with DnaK. We found that His₆ tags uniquely promote binding to additional sites in the substrate binding domain at the C-terminus of DnaK. Other C-terminal tags attached to J-domains, even peptides known to interact with DnaK, do not produce the same effects. Expression of C-terminally modified DnaJ1 or DnaJ2 J-domains in mycobacterial cells suppresses chaperone activity following proteotoxic stress, which is exaggerated in the presence of a small-molecule DnaK inhibitor. Hence, this work uncovers genetically encodable J-protein variants that may be used to study chaperone–cofactor interactions in other organisms.     

Structural Communication between the E. coli Chaperones DnaK and Hsp90

The 70 kDa and 90 kDa heat shock proteins Hsp70 and Hsp90 are two abundant and highly conserved ATP-dependent molecular chaperones that participate in the maintenance of cellular homeostasis. In Escherichia coli, Hsp90 (Hsp90Ec) and Hsp70 (DnaK) directly interact and collaborate in protein remodeling. Previous work has produced a model of the direct interaction of both chaperones. The locations of the residues involved have been confirmed and the model has been validated. In this study, we investigate the allosteric communication between Hsp90Ec and DnaK and how the chaperones couple their conformational cycles. Using elastic network models (ENM), normal mode analysis (NMA), and a structural perturbation method (SPM) of asymmetric and symmetric DnaK-Hsp90Ec, we extract biologically relevant vibrations and identify residues involved in allosteric signaling. When one DnaK is bound, the dominant normal modes favor biological motions that orient a substrate protein bound to DnaK within the substrate/client binding site of Hsp90Ec and release the substrate from the DnaK substrate binding domain. The presence of one DnaK molecule stabilizes the entire Hsp90Ec protomer to which it is bound. Conversely, the symmetric model of DnaK binding results in steric clashes of DnaK molecules and suggests that the Hsp90Ec and DnaK chaperone cycles operate independently. Together, this data supports an asymmetric binding of DnaK to Hsp90Ec.     

Rational Design of Novel Peptidic DnaK Ligands

The hsp70 chaperone DnaK from E. coli plays a major role in cellular stress response and is involved in assisted protein folding in vivo. By screening a combinatorial peptide library, we identified several DnaK‐specific peptide ligands with nanomolar affinities, which are able to inhibit the secondary amide peptide bond cis/trans isomerase (APIase) activity of DnaK, as well as DnaK/DnaJ/GrpE‐assisted refolding of firefly luciferase. Our designed DnaK inhibitors have the capability to penetrate E. coli cells and feature a high protease resistance. Once inside the cell, they physically target DnaK. NMR‐based ¹H/¹⁵N‐HSQC experiments furthermore confirmed that the designed peptidic ligands all bind in an identical manner to the conventional peptide‐binding site of DnaK. The subsequent blocking of DnaK function apparently results in the observed antibacterial effects on E. coli cells, with minimum inhibitory concentrations in the range of 100 μM .     

Regulation of ClC-2 Chloride Channel Proteostasis by Molecular Chaperones: Correction of Leukodystrophy-Associated Defect

The ClC-2 channel plays a critical role in maintaining ion homeostasis in the brain and the testis. Loss-of-function mutations in the ClC-2-encoding human CLCN2 gene are linked to the white matter disease leukodystrophy. Clcn2-deficient mice display neuronal myelin vacuolation and testicular degeneration. Leukodystrophy-causing ClC-2 mutant channels are associated with anomalous proteostasis manifesting enhanced endoplasmic reticulum (ER)-associated degradation. The molecular nature of the ER quality control system for ClC-2 protein remains elusive. In mouse testicular tissues and Leydig cells, we demonstrated that endogenous ClC-2 co-existed in the same protein complex with the molecular chaperones heat shock protein 90β (Hsp90β) and heat shock cognate protein (Hsc70), as well as the associated co-chaperones Hsp70/Hsp90 organizing protein (HOP), activator of Hsp90 ATPase homolog 1 (Aha1), and FK506-binding protein 8 (FKBP8). Further biochemical analyses revealed that the Hsp90β-Hsc70 chaperone/co-chaperone system promoted mouse and human ClC-2 protein biogenesis. FKBP8 additionally facilitated membrane trafficking of ClC-2 channels. Interestingly, treatment with the Hsp90-targeting small molecule 17-allylamino-17-demethoxygeldanamycin (17-AAG) substantially boosted ClC-2 protein expression. Also, 17-AAG effectively increased both total and cell surface protein levels of leukodystrophy-causing loss-of-function ClC-2 mutant channels. Our findings highlight the therapeutic potential of 17-AAG in correcting anomalous ClC-2 proteostasis associated with leukodystrophy.     

Influence of Substitutions in the Binding Motif of Proline-Rich Antimicrobial Peptide ARV-1502 on 70S Ribosome Binding and Antimicrobial Activity

Proline-rich antimicrobial peptides (PrAMPs) are promising candidates to treat bacterial infections. The designer peptide ARV-1502 exhibits strong antimicrobial effects against Enterobacteriaceae both in vitro and in vivo. Since the inhibitory effects of ARV-1502 reported for the 70 kDa heat-shock protein DnaK do not fully explain the antimicrobial activity of its 176 substituted analogs, we further studied their effect on the bacterial 70S ribosome of Escherichia coli, a known target of PrAMPs. ARV-1502 analogues, substituted in positions 3, 4, and 8 to 12 (underlined) of the binding motif D³KPRPYLPRP¹² with aspartic acid, lysine, serine, phenylalanine or leucine, were tested in a competitive fluorescence polarization (FP) binding screening assay using 5(6)-carboxyfluorescein-labeled (Cf-) ARV-1502 and the 70S ribosome isolated from E. coli BW25113. While their effect on ribosomal protein expression was studied for green fluorescent protein (GFP) in a cell-free expression system (in vitro translation), the importance of known PrAMP transporters SbmA and MdtM was investigated using E. coli BW25113 and the corresponding knockout mutants. The dissociation constant (K_d) of 201 ± 16 nmol/L obtained for Cf-ARV-1502 suggests strong binding to the E. coli 70S ribosome. An inhibitory binding assay indicated that the binding site overlaps with those of other PrAMPs including Onc112 and pyrrhocoricin as well as the non-peptidic antibiotics erythromycin and chloramphenicol. All these drugs and drug candidates bind to the exit-tunnel of the 70S ribosome. Substitutions of the C-terminal fragment of the binding motif YLPRP reduced binding. At the same time, inhibition of GFP expression increased with net peptide charge. Interestingly, the MIC values of wild-type and ΔsbmA and ΔmdtM knockout mutants indicated that substitutions in the ribosomal binding motif altered also the bacterial uptake, which was generally improved by incorporation of hydrophobic residues. In conclusion, most substituted ARV-1502 analogs bound weaker to the 70S ribosome than ARV-1502 underlining the importance of the YLPRP binding motif. The weaker ribosomal binding correlated well with decreased antimicrobial activity in vitro. Substituted ARV-1502 analogs with a higher level of hydrophobicity or positive net charge improved the ribosome binding, inhibition of translation, and bacterial uptake.     

Modeling and Docking Studies on Novel Mutants (K71L and T204V) of the ATPase Domain of Human Heat Shock 70 kDa Protein 1

The purpose of exploring protein interactions between human adenovirus and heat shock protein 70 is to exploit a potentially synergistic interaction to enhance anti-tumoral efficacy and decrease toxicity in cancer treatment. However, the protein interaction of Hsp70 with E1A32 kDa of human adenovirus serotype 5 remains to be elucidated. In this study, two residues of ATPase domain of human heat shock 70 kDa protein 1 (PDB: 1 HJO) were mutated. 3D mutant models (K71L and T204V) using PyMol software were then constructed. The structures were evaluated by PROCHECK, ProQ, ERRAT, Verify 3D and ProSA modules. All evidence suggests that all protein models are acceptable and of good quality. The E1A32 kDa motif was retrieved from UniProt ({"type":"entrez-protein","attrs":{"text":"P03255","term_id":"119020","term_text":"P03255"}}P03255), as well as subjected to docking interaction with NBD, K71L and T204V, using the Autodock 4.2 program. The best lowest binding energy value of −9.09 kcal/mol was selected for novel T204V. Moreover, the protein-ligand complex structures were validated by RMSD, RMSF, hydrogen bonds and salt bridge analysis. This revealed that the T204V-E1A32 kDa motif complex was the most stable among all three complex structures. This study provides information about the interaction between Hsp70 and the E1A32 kDa motif, which emphasizes future perspectives to design rational drugs and vaccines in cancer therapy.     

Tracking the Interplay between Bound Peptide and the Lid Domain of DnaK,Using Molecular Dynamics

Hsp70 chaperones consist of two functional domains: the 44 kDa Nucleotide Binding Domain (NBD), that binds and hydrolyses ATP, and the 26 kDa Substrate Binding Domain (SBD), which binds unfolded proteins and reactivates them, utilizing energy obtained from nucleotide hydrolysis. The structure of the SBD of the bacterial Hsp70, DnaK, consists of two sub-domains: A β-sandwich part containing the hydrophobic cavity to which the hepta-peptide NRLLLTG (NR) is bound, and a segment made of 5 α-helices, called the “lid” that caps the top of the β-sandwich domain. In the present study we used the Escherichia coli Hsp70, DnaK, as a model for Hsp70 proteins, focusing on its SBD domain, examining the changes in the lid conformation. We deliberately decoupled the NBD from the SBD, limiting the study to the structure of the SBD section, with an emphasis on the interaction between the charges of the peptide with the residues located in the lid. Molecular dynamics simulations of the complex revealed significant mobility within the lid structure; as the structure was released from the forces operating during the crystallization process, the two terminal helices established a contact with the positive charge at the tip of the peptide. This contact is manifested only in the presence of electrostatic attraction. The observed internal motions within the lid provide a molecular role for the function of this sub-domain during the reaction cycle of Hsp 70 chaperones.     

Characterization of CaHsp70-1, a Pepper Heat-Shock Protein Gene in Response to Heat Stress and Some Regulation Exogenous Substances in Capsicum annuum L.

Pepper (Capsicum annuum L.) is sensitive to heat stress (HS). Heat shock proteins 70 (Hsp70s) play a crucial role in protecting plant cells against HS and control varies characters in different plants. However, CaHsp70-1 gene was not well characterized in pepper. In this study, CaHsp70-1 was cloned from the pepper thermotolerant line R9, which encoded a protein of 652 amino acids, with a molecular weight of 71.54 kDa and an isoelectric point of 5.20. CaHsp70-1 belongs to the cytosolic Hsp70 subgroup, and best matched with tomato SlHsp70. CaHsp70-1 was highly induced in root, stem, leaf and flower in R9 with HS treatment (40 °C for 2 h). In both thermosensitive line B6 and thermotolerant line R9, CaHsp70-1 significantly increased after 0.5 h of HS (40 °C), and maintained in a higher level after 4 h HS. The expression of CaHsp70-1 induced by CaCl₂, H₂O₂ and putrescine (Put) under HS were difference between B6 and R9 lines. The different expression patterns may be related to the differences in promoters of CaHsp70-1 from the two lines. These results suggest that CaHsp70-1 as a member of cytosolic Hsp70 subgroup, may be involved in HS defense response via a signal transduction pathway contained Ca²⁺, H₂O₂ and Put.     

Chaperone Hsp70 (HSPA1) Is Involved in the Molecular Mechanisms of Sleep Cycle Integration

The molecular mechanisms of sleep cycle integration at the beginning and the end of the inactive period are not clear. Sleep cycles with a predominance of deep slow-wave sleep (SWS) seem to be associated with accelerated protein synthesis in the brain. The inducible Hsp70 chaperone corrects protein conformational changes and has protective properties. This research explores (1) whether the Hspa1 gene encoding Hsp70 protein activates during the daily rapid-eye-movement sleep (REMS) maximum, and (2) whether a lower daily deep SWS maximum affects the Hspa1 expression level during the subsequent REMS. Combining polysomnography in male Wistar rats, RT-qPCR, and Western blotting, we reveal a three-fold Hspa1 upregulation in the nucleus reticularis pontis oralis, which regulates REMS. Hspa1 expression increases during the daily REMS maximum, 5–7 h after the natural peak of deep SWS. Using short-term selective REMS deprivation, we demonstrate that REMS rebound after deprivation exceeds the natural daily maximum, but it is not accompanied by Hspa1 upregulation. The results suggest that a high proportion of deep SWS, usually observed after sleep onset, is a necessary condition for Hspa1 upregulation during subsequent REMS. The data obtained can inform the understanding of the molecular mechanisms integrating SWS and REMS and key biological function(s) of sleep.     

Protein design to understand peptide ligand recognition by tetratricopeptide repeat proteins

Protein design aims to understand the fundamentals of protein structure by creating novel proteins with pre-specified folds. An equally important goal is to understand protein function by creating novel proteins with pre-specified activities. Here we describe the design and characterization of a tetratricopeptide (TPR) protein, which binds to the C-terminal peptide of the eukaryotic chaperone Hsp90. The design emphasizes the importance of both direct, short-range protein-peptide interactions and of long-range electrostatic optimization. We demonstrate that the designed protein binds specifically to the desired peptide and discriminates between it and the similar C-terminal peptide of Hsp70.     

Fine tuning of a biological machine: DnaK gains improved chaperone activity by altered allosteric communication and substrate binding

DnaK is a member of the Hsp70 family of molecular chaperones. This molecular machine couples the binding and hydrolysis of ATP to binding and release of substrate proteins. The switches that are involved in allosteric communication within this multidomain protein are mostly unknown. Previous insights were largely obtained by mutants, which displayed either wild-type activity or reduced folding assistance of substrate proteins. With a directed evolution approach for improved folding assistance we selected a DnaK variant characterized by a glycine to alanine substitution at position 384 (G384A); this resulted in a 2.5-fold higher chaperone activity in an in vitro DnaK-assisted firefly luciferase refolding assay. Quantitative biochemical characterization revealed several changes of key kinetic parameters compared to the wild type. Most pronounced is a 13-fold reduced rate constant for substrate release in the ATP-bound state, which we assume, in conjunction with the resulting increase in substrate affinity, to be related to improved chaperone activity. As the underlying mechanistic reason for this change we propose an altered interface of allosteric communication of mutant G384A, which is notably located at a hinge position between nucleotide and substrate binding domain.     

NMR chemical shift perturbation study of the N-terminal domain of Hsp90 upon binding of ADP,AMP-PNP,geldanamycin, and radicicol

Hsp90 is one of the most abundant chaperone proteins in the cytosol. In an ATP-dependent manner it plays an essential role in the folding and activation of a range of client proteins involved in signal transduction and cell cycle regulation. We used NMR shift perturbation experiments to obtain information on the structural implications of the binding of AMP-PNP (adenylyl-imidodiphosphate-a non-hydrolysable ATP analogue), ADP and the inhibitors radicicol and geldanamycin. Analysis of (1)H,(15)N correlation spectra showed a specific pattern of chemical shift perturbations at N210 (ATP binding domain of Hsp90, residues 1-210) upon ligand binding. This can be interpreted qualitatively either as a consequence of direct ligand interactions or of ligand-induced conformational changes within the protein. All ligands show specific interactions in the binding site, which is known from the crystal structure of the N-terminal domain of Hsp90. For AMP-PNP and ADP, additional shift perturbations of residues outside the binding pocket were observed and can be regarded as a result of conformational rearrangement upon binding. According to the crystal structures, these regions are the first alpha-helix and the "ATP-lid" ranging from amino acids 85 to 110. The N-terminal domain is therefore not a passive nucleotide-binding site, as suggested by X-ray crystallography, but responds to the binding of ATP in a dynamic way with specific structural changes required for the progression of the ATPase cycle.     

Structural Basis for Design of New Purine-Based Inhibitors Targeting the Hydrophobic Binding Pocket of Hsp90

Inhibition of the molecular chaperone heat shock protein 90 (Hsp90) represents a promising approach for cancer treatment. BIIB021 is a highly potent Hsp90 inhibitor with remarkable anticancer activity; however, its clinical application is limited by lack of potency and response. In this study, we aimed to investigate the impact of replacing the hydrophobic moiety of BIIB021, 4-methoxy-3,5-dimethylpyridine, with various five-membered ring structures on the binding to Hsp90. A focused array of N⁷/N⁹-substituted purines, featuring aromatic and non-aromatic rings, was designed, considering the size of hydrophobic pocket B in Hsp90 to obtain insights into their binding modes within the ATP binding site of Hsp90 in terms of π–π stacking interactions in pocket B as well as outer α-helix 4 configurations. The target molecules were synthesized and evaluated for their Hsp90α inhibitory activity in cell-free assays. Among the tested compounds, the isoxazole derivatives 6b and 6c, and the sole six-membered derivative 14 showed favorable Hsp90α inhibitory activity, with IC₅₀ values of 1.76 µM, 0.203 µM, and 1.00 µM, respectively. Furthermore, compound 14 elicited promising anticancer activity against MCF-7, SK-BR-3, and HCT116 cell lines. The X-ray structures of compounds 4b, 6b, 6c, 8, and 14 bound to the N-terminal domain of Hsp90 were determined in order to understand the obtained results and to acquire additional structural insights, which might enable further optimization of BIIB021.     

Anti-Trypanosoma cruzi Activity of Metabolism Modifier Compounds

Chagas disease is caused by the protozoan parasite Trypanosoma cruzi and affects over 6 million people worldwide. Development of new drugs to treat this disease remains a priority since those currently available have variable efficacy and frequent adverse effects, especially during the long regimens required for treating the chronic stage of the disease. T. cruzi modulates the host cell-metabolism to accommodate the cell cytosol into a favorable growth environment and acquire nutrients for its multiplication. In this study we evaluated the specific anti-T. cruzi activity of nine bio-energetic modulator compounds. Notably, we identified that 17-DMAG, which targets the ATP-binding site of heat shock protein 90 (Hsp90), has a very high (sub-micromolar range) selective inhibition of the parasite growth. This inhibitory effect was also highly potent (IC₅₀ = 0.27 μmol L⁻¹) against the amastigote intracellular replicative stage of the parasite. Moreover, molecular docking results suggest that 17-DMAG may bind T. cruzi Hsp90 homologue Hsp83 with good affinity. Evaluation in a mouse model of chronic T. cruzi infection did not show parasite growth inhibition, highlighting the difficulties encountered when going from in vitro assays onto preclinical drug developmental stages.     

DnaJC7 in Amyotrophic Lateral Sclerosis

Protein misfolding is a common basis of many neurodegenerative diseases including amyotrophic lateral sclerosis (ALS). Misfolded proteins, such as TDP-43, FUS, Matrin3, and SOD1, mislocalize and form the hallmark cytoplasmic and nuclear inclusions in neurons of ALS patients. Cellular protein quality control prevents protein misfolding under normal conditions and, particularly, when cells experience protein folding stress due to the fact of increased levels of reactive oxygen species, genetic mutations, or aging. Molecular chaperones can prevent protein misfolding, refold misfolded proteins, or triage misfolded proteins for degradation by the ubiquitin–proteasome system or autophagy. DnaJC7 is an evolutionarily conserved molecular chaperone that contains both a J-domain for the interaction with Hsp70s and tetratricopeptide domains for interaction with Hsp90, thus joining these two major chaperones’ machines. Genetic analyses reveal that pathogenic variants in the gene encoding DnaJC7 cause familial and sporadic ALS. Yet, the underlying ALS-associated molecular pathophysiology and many basic features of DnaJC7 function remain largely unexplored. Here, we review aspects of DnaJC7 expression, interaction, and function to propose a loss-of-function mechanism by which pathogenic variants in DNAJC7 contribute to defects in DnaJC7-mediated chaperoning that might ultimately contribute to neurodegeneration in ALS.     

HSP70-Homolog DnaK of Pseudomonas aeruginosa Increases the Production of IL-27 through Expression of EBI3 via TLR4-Dependent NF-κB and TLR4-Independent Akt Signaling

IL-27, a heterodimeric cytokine composed of the p28 subunit and Epstein–Barr virus-induced gene 3 (EBI3), acts as a potent immunosuppressant and thus limits pathogenic inflammatory responses. IL-27 is upregulated upon Pseudomonas aeruginosa infection in septic mice, increasing susceptibility to the infection and decreasing clearance of the pathogen. However, it remains unclear which P. aeruginosa-derived molecules promote production of IL-27. In this study, we explored the mechanism by which P. aeruginosa DnaK, a heat shock protein 70-like protein, induces EBI3 expression, thereby promoting production of IL-27. Upregulation of EBI3 expression did not lead to an increase in IL-35, which consists of the p35 subunit and EBI3. The IL-27 production in response to DnaK was biologically active, as reflected by stimulation of IL-10 production. DnaK-mediated expression of EBI3 was driven by two distinct signaling pathways, NF-κB and Akt. However, NF-κB is linked to TLR4-associated signaling pathways, whereas Akt is not. Taken together, our results reveal that P. aeruginosa DnaK potently upregulates EBI3 expression, which in turn drives production of the prominent anti-inflammatory cytokine IL-27, as a consequence of TLR4-dependent activation of NF-κB and TLR4-independent activation of the Akt signaling pathway.