DnaK ATPase-BD E.Coli

What is the fundamental role of DnaK's ATPase activity in E. coli?

DnaK, a member of the Hsp70 (Heat Shock Protein 70) family in E. coli, relies on its ATPase activity to power its chaperone functions. This protein was originally identified through mutations that blocked lambda phage DNA replication and has since been recognized as essential for E. coli viability. DnaK possesses a weak, DNA-independent ATPase activity that hydrolyzes ATP to ADP and inorganic phosphate (Pi) . This enzymatic function drives cyclic conformational changes that enable DnaK to bind and release client proteins, facilitating protein folding, preventing aggregation, and rescuing misfolded proteins. The ATPase activity serves as the molecular motor that powers DnaK's ability to maintain proteostasis in the bacterial cell.

How is DnaK's ATPase activity regulated by co-chaperones in E. coli?

DnaK's intrinsic ATPase activity is relatively weak but becomes significantly enhanced through interactions with co-chaperones. The primary co-chaperone, DnaJ (Hsp40), substantially stimulates DnaK's ATPase activity by accelerating the conversion of ATP to ADP . This stimulation triggers conformational changes in DnaK that strengthen its interaction with substrate proteins. E. coli possesses two additional DnaJ homologs—CbpA and DjlA—that can also interact with DnaK and stimulate its ATPase activity, though with different efficiencies . The coordinated regulation of DnaK's ATPase activity by these co-chaperones enables fine-tuning of the chaperone system's response to different cellular conditions and protein folding needs.

What experimental evidence establishes the link between DnaK's gene and its ATPase activity?

The connection between the dnaK gene and the observed ATPase activity has been established through genetic and biochemical approaches. The primary evidence comes from studies showing that the dnaK756 mutation results in an ATPase activity with altered physical properties . This mutation-function relationship demonstrates that changes in the dnaK gene directly affect ATPase properties, confirming that the gene encodes the protein responsible for this enzymatic function. Additionally, purified wild-type DnaK protein exhibits ATP hydrolysis activity resulting in ADP and Pi production in vitro, providing direct biochemical evidence for this functionality .

How can researchers experimentally assess the redundancy among DnaJ homologs in the DnaK chaperone system?

To evaluate functional redundancy among DnaJ, CbpA, and DjlA in the DnaK chaperone system, researchers can employ several methodological approaches:

• Genetic analysis using single, double, and triple mutants of dnaJ, cbpA, and djlA to assess growth phenotypes under various stress conditions.

• In vivo protein aggregation systems that simultaneously examine multiple cellular proteins' aggregation states in these mutant backgrounds .

• In vitro disaggregation assays using preheated extracts from the triple mutant (dnaJ-cbpA-djlA) complemented with purified DnaJ homologs .

• Comparative proteome analysis of aggregated proteins in different mutant backgrounds to identify protein subsets preferentially dependent on specific co-chaperones.

• Temperature-sensitivity assays comparing wild-type and mutant strains, which revealed that "both dnaJ-cbpA and dnaJ-djlA double mutants are temperature sensitive, while the cbpA-djlA double mutant is not" .

These approaches have demonstrated that CbpA and DjlA can partially substitute for DnaJ in DnaK-mediated disaggregation, though complete replacement of DnaJ's function requires both homologs .

What experimental evidence supports functional substitution among DnaJ homologs?

Multiple lines of experimental evidence demonstrate that DnaJ homologs can partially substitute for each other in supporting DnaK function:

These findings establish that while DnaJ remains the primary co-chaperone, the DnaJ homologs provide functional redundancy that likely contributes to the robustness of the cellular protein quality control system.

How do lambda phage proteins interact with DnaK's ATPase function?

Lambda phage proteins have been shown to specifically modulate DnaK's ATPase function through direct protein-protein interactions. Experimental evidence indicates that:

• The lambda O and P replication proteins interact in vitro with DnaK protein .

• Lambda P protein inhibits the ATPase activity of DnaK .

• Conversely, lambda O protein stimulates DnaK's ATPase activity .

These interactions suggest a mechanism by which lambda phage can regulate host DnaK function during infection, potentially redirecting the chaperone's activity to support viral replication. These findings also provide insight into how DnaK's ATPase activity can be modulated by specific protein interactions, which may inform strategies for therapeutic targeting of this enzyme. The contrasting effects of the O and P proteins (stimulation versus inhibition) demonstrate the complex regulation possible through protein-protein interactions with DnaK.

What are the recommended protocols for measuring DnaK's ATPase activity in vitro?

Researchers can measure DnaK's ATPase activity in vitro using several established methods:

• Colorimetric phosphate detection: Using malachite green or other phosphate-binding reagents to quantify released inorganic phosphate.

• Radioactive ATP hydrolysis assays: Tracking the conversion of [γ-³²P]ATP to ADP and ³²P-labeled inorganic phosphate.

• Coupled enzyme assays: Linking ATP hydrolysis to NADH oxidation through pyruvate kinase and lactate dehydrogenase, measurable by spectrophotometry.

For accurate measurement, reactions should typically include:

• Purified DnaK (0.5-2 μM)

• ATP (50-200 μM)

• Buffer containing Tris-HCl (pH 7.5-8.0), KCl (100-150 mM), and MgCl₂ (5-10 mM)

• Optional: DnaJ or other co-chaperones to measure stimulated activity

When assessing modulation by other factors (co-chaperones, substrate proteins, inhibitors), appropriate controls should include DnaK alone and heat-inactivated DnaK to establish baseline and non-specific hydrolysis rates. The search results indicate that DnaK's ATPase activity can be significantly influenced by other proteins, making careful experimental design essential .

How can researchers distinguish between intrinsic and stimulated ATPase activities of DnaK?

To differentiate between DnaK's intrinsic ATPase activity and its stimulated activity, researchers should implement the following methodological approaches:

• Sequential addition experiments: Measure baseline ATPase activity with DnaK alone, then add co-chaperones (DnaJ, CbpA, or DjlA) to the same reaction and monitor the change in hydrolysis rate. The search results indicate that DnaJ significantly enhances DnaK's ATPase activity .

• Concentration-dependent studies: Perform ATPase assays with DnaK at a fixed concentration while varying the concentration of co-chaperones to establish dose-response relationships.

• Mutational analysis: Compare ATPase activities of wild-type DnaK with mutant variants defective in co-chaperone interactions but retaining intrinsic ATPase function.

• Inhibition studies: Use peptides or small molecules that specifically block the interaction between DnaK and its co-chaperones to isolate intrinsic activity.

• Kinetic analysis: Determine Km and kcat values for ATP hydrolysis with and without co-chaperones to characterize changes in enzyme kinetics.

The difference between measured rates with and without co-chaperones provides quantitative assessment of stimulation, which can reveal mechanistic insights into how co-chaperones enhance DnaK's enzymatic function.

What factors can influence DnaK's ATPase assays and how should they be controlled?

Several factors can significantly influence DnaK's ATPase activity measurements and must be carefully controlled:

• Temperature effects: DnaK's activity is temperature-dependent, with disaggregation studies typically performed at physiologically relevant temperatures (37-42°C) . Maintain precise temperature control during assays.

• Co-chaperone concentrations: DnaJ, CbpA, and DjlA stimulate DnaK's ATPase activity to different degrees . Use defined concentrations and include appropriate controls when assessing their effects.

• Substrate proteins: Client proteins can modulate DnaK's ATPase activity. The search results show that "DnaK associates more with AMR-conferring mutant RNA polymerase (RNAP) than with wild-type RNAP" , suggesting differential interactions with mutant proteins.

• Buffer composition: pH, ionic strength, and divalent cation concentrations significantly affect ATPase activity. Use consistent buffer conditions and include proper controls when comparing across experiments.

• Contaminating ATPases: Ensure high purity of DnaK preparations to avoid interference from contaminating ATPases. Include controls with heat-inactivated DnaK.

• ATP concentration: Use consistent ATP concentrations across experiments, typically at or above the Km value for DnaK.

• Protein phosphorylation state: DnaK undergoes phosphorylation that may affect its activity . Use defined phosphorylation states when comparing activities.

Rigorous control of these factors is essential for obtaining reproducible and physiologically relevant measurements of DnaK's ATPase activity.

How can researchers experimentally track protein disaggregation mediated by the DnaK system?

Researchers can track DnaK-mediated protein disaggregation using several methodological approaches:

• In vivo protein aggregation systems: The search results describe a system that "makes it possible to simultaneously examine the aggregated state of a large number of cellular proteins" . This involves inducing protein aggregation (typically by heat shock), then monitoring the disappearance of aggregates and reappearance of soluble proteins over time.

• Specific protein tracking: As demonstrated with homoserine transsuccinylase (HTS), researchers can selectively track a single protein's aggregation and disaggregation by:

  • Inducing aggregation through heat shock

  • Blocking new protein synthesis (using methionine to repress HTS synthesis)

  • Monitoring the disappearance of the protein from the aggregated fraction and its reappearance in the soluble fraction

• Fractionation techniques: Separating soluble and insoluble cellular fractions at different time points after heat shock to quantify changes in protein distribution.

• Fluorescence-based approaches: Using fluorescently tagged proteins to visualize aggregation and disaggregation in real-time.

These approaches allow researchers to distinguish between proteins that are renatured versus those that are degraded following disaggregation, with the search results indicating that "most of the aggregated HTS was renatured and not proteolyzed" .

What methodological approaches can distinguish between protein renaturation and degradation after disaggregation?

To determine whether disaggregated proteins are renatured or degraded, researchers should employ the following methodological strategies:

• Protein synthesis inhibition coupled with tracking: The search results describe an elegant experimental system using homoserine transsuccinylase (HTS) where:

  • HTS synthesis is repressed by adding methionine

  • Aggregation is induced by heat shock at 46°C

  • After shifting back to lower temperature, the fate of pre-existing HTS is monitored in both aggregated and soluble fractions

  This approach revealed that "most of the aggregated HTS was renatured and not proteolyzed" .

• Activity assays: Measuring the enzymatic activity of model proteins before heat shock, immediately after (when aggregated), and following recovery to quantify functional restoration.

• Pulse-chase experiments: Using radioactive labeling to track the fate of a specific cohort of proteins through aggregation and disaggregation.

• Protease inhibition studies: Comparing disaggregation patterns with and without protease inhibitors to determine the contribution of proteolytic degradation.

• Quantitative proteomics: Using stable isotope labeling to distinguish between newly synthesized proteins and those that survived the aggregation-disaggregation cycle.

These methodologies allow researchers to quantitatively assess whether the DnaK system primarily functions to restore protein function or to target aggregated proteins for degradation.

How do mutations in the DnaK system components affect protein disaggregation capacity?

Mutations in DnaK system components significantly impact protein disaggregation capacity, as revealed by several experimental approaches:

• Single and multiple mutant comparisons: Studies show that "following heat shock, aggregation of proteins in dnaK mutants is more pronounced than it is in the wild type" . Additionally, "in a dnaK mutant the aggregates are not dissolved over time" , demonstrating DnaK's essential role in disaggregation.

• Co-chaperone redundancy analysis: In dnaJ mutants, "both CbpA and DjlA are required for efficient protein dissaggregation at 42°C" , indicating partial functional redundancy among co-chaperones.

• Temperature-sensitive phenotypes: Double mutants (dnaJ-cbpA and dnaJ-djlA) show temperature sensitivity that correlates with their disaggregation defects, while the cbpA-djlA double mutant retains temperature resistance .

• DnaK expression levels: Interestingly, "the levels of DnaK in the mutants defective in disaggregation were higher than the levels in the wild type" , suggesting compensatory upregulation that is insufficient to overcome the loss of co-chaperones.

• Triple mutant analysis: The dnaJ-cbpA-djlA triple mutant shows the most severe disaggregation defects despite having the highest DnaK levels (three-fold higher than wild type) , demonstrating the essential nature of co-chaperones in the disaggregation process.

These findings highlight the complex interplay between DnaK and its co-chaperones in protein disaggregation and provide methodological frameworks for studying chaperone system mutations.

What experimental evidence links DnaK to antimicrobial resistance in bacteria?

Several lines of experimental evidence establish DnaK's role in supporting antimicrobial resistance (AMR):

• Protein interaction studies: Research demonstrates that "DnaK associates with many drug targets and that DnaK associates more with AMR-conferring mutant RNA polymerase (RNAP) than with wild-type RNAP" . This preferential association suggests DnaK stabilizes mutant forms of drug targets.

• Frequency-of-resistance (FOR) studies: Investigations reveal that "the DnaK system of chaperones supports AMR in antimicrobial targets in mycobacteria, including RNAP and the ribosome" .

• Cross-species validation: Studies across bacterial species show that "overexpression of the groE operon in Escherichia coli increased the fitness of strains that had accumulated mutations" and "deletion of E. coli DnaK slowed resistance evolution to tetracycline" .

• Client protein identification: DnaK associates with multiple antimicrobial targets and proteins in pathways inhibited by antimicrobials, including those involved in resistance to isoniazid, ethionamide, fluoroquinolones, rifampin, and capreomycin .

• Mutational burden response: When error-prone DNA polymerase was expressed in Salmonella, "levels of DnaK and GroEL increased in lineages with high mutational burdens" , suggesting chaperones help compensate for potentially destabilizing mutations.

This evidence collectively establishes DnaK as a critical factor in supporting AMR through stabilization of mutant proteins that confer resistance.

How can researchers experimentally assess DnaK's contribution to the fitness of resistant bacterial strains?

To experimentally assess DnaK's contribution to the fitness of resistant bacteria, researchers can employ these methodological approaches:

• Genetic depletion systems: Create conditional DnaK depletion strains in resistant bacterial backgrounds to measure how DnaK reduction affects growth rates, survival under stress, and maintenance of resistance.

• Competitive fitness assays: Conduct direct competition experiments between wild-type and DnaK-depleted resistant strains to quantify fitness differences.

• SILAC proteomics: Use Stable Isotope Labeling with Amino acids in Cell culture to identify and quantify DnaK's differential association with wild-type versus resistant target proteins .

• Solubility profiling: Compare the solubility of resistance-conferring proteins (like mutant RNA polymerase) in the presence and absence of functional DnaK to determine if DnaK prevents aggregation of these potentially unstable mutant proteins.

• Evolution experiments: Perform adaptive laboratory evolution under antibiotic selection with varying levels of DnaK expression to track resistance acquisition rates and pathways.

• Thermal stability assays: Compare thermal denaturation profiles of resistance proteins with and without functional DnaK to assess its stabilizing effects.

• Minimum inhibitory concentration (MIC) testing: Measure changes in antibiotic susceptibility when DnaK function is altered to quantify its direct impact on resistance levels.

These approaches can provide quantitative assessment of how DnaK contributes to the fitness of bacteria harboring resistance mutations.

What methodological approaches can be used to identify potential DnaK inhibitors for combating antimicrobial resistance?

Researchers can employ several methodological approaches to identify and develop DnaK inhibitors as potential adjuvants for combating antimicrobial resistance:

• High-throughput ATPase assays: Screen compound libraries for molecules that inhibit DnaK's ATPase activity using colorimetric or fluorescent detection of ATP hydrolysis.

• Structure-based drug design: Utilize crystal structures of DnaK's ATPase domain to design compounds that compete with ATP binding or allosterically inhibit ATPase function.

• Fragment-based screening: Identify small molecular fragments that bind to DnaK and can be elaborated into more potent inhibitors.

• Protein-protein interaction disruptors: Screen for compounds that disrupt critical interactions between DnaK and its co-chaperones or client proteins, particularly those involved in antimicrobial resistance.

• Phenotypic screening: Test compounds for their ability to sensitize resistant bacteria to antibiotics when DnaK is targeted.

• Combination studies: Evaluate potential DnaK inhibitors in combination with conventional antibiotics against resistant bacterial strains to identify synergistic interactions.

• Resistance development monitoring: Assess how DnaK inhibition affects the frequency and mechanisms of resistance development to various antibiotics.

The search results highlight chaperones as "potential targets for drugs to overcome AMR in mycobacteria, including M. tuberculosis, as well as in other pathogens" , supporting this approach as a viable strategy for addressing antimicrobial resistance.

How does DnaK phosphorylation affect its ATPase function and chaperone activity?

DnaK undergoes phosphorylation that may significantly impact its functional properties. The search results indicate that "The dnaK protein is phosphorylated in vitro and in vivo, probably as a result of an autophosphorylation reaction" . To investigate this post-translational modification's effects on DnaK's ATPase and chaperone functions, researchers should employ these methodological approaches:

• Phosphorylation site mapping: Use mass spectrometry to identify specific residues that undergo phosphorylation in vivo and in vitro.

• Phosphomimetic mutants: Create DnaK variants with mutations at phosphorylation sites (e.g., serine/threonine to aspartate) to mimic constitutive phosphorylation, then assess their ATPase activity and chaperone function.

• Phospho-null mutants: Generate non-phosphorylatable variants (serine/threonine to alanine) to examine the consequences of preventing phosphorylation.

• Controlled phosphorylation assays: Develop protocols for generating homogeneously phosphorylated or non-phosphorylated DnaK preparations for comparative functional analysis.

• Phosphorylation kinetics: Analyze how rapidly phosphorylation occurs and whether it correlates with changes in ATPase activity.

• Structural studies: Use X-ray crystallography or cryo-EM to determine how phosphorylation affects DnaK's conformation.

Understanding phosphorylation's role in modulating DnaK function could provide insights into how cells fine-tune their chaperone systems under different stress conditions and potentially reveal new regulatory mechanisms.

What techniques can be used to study conformational changes in DnaK during its ATPase cycle?

Studying the conformational dynamics of DnaK during its ATPase cycle requires sophisticated biophysical approaches:

• FRET (Förster Resonance Energy Transfer): Label different domains of DnaK with fluorophore pairs to monitor distance changes between domains during ATP binding, hydrolysis, and ADP release.

• HDX-MS (Hydrogen-Deuterium Exchange Mass Spectrometry): Map regions of DnaK that undergo changes in solvent accessibility in different nucleotide-bound states.

• Cryo-EM (Cryo-Electron Microscopy): Capture DnaK in different conformational states through vitrification at various stages of the ATPase cycle.

• NMR (Nuclear Magnetic Resonance) spectroscopy: Particularly useful for studying dynamics of specific DnaK domains in solution with different nucleotides.

• EPR (Electron Paramagnetic Resonance) with site-directed spin labeling: Measure distances between specific sites in different DnaK conformations.

• Single-molecule techniques: Use optical tweezers or TIRF microscopy to observe individual DnaK molecules as they cycle through conformational states.

• Molecular dynamics simulations: Complement experimental approaches with computational modeling of DnaK's conformational changes at atomic resolution.

These techniques can provide mechanistic insights into how ATP binding and hydrolysis drive the structural rearrangements that enable DnaK's chaperone functions, including client protein binding and release cycles.

How can systems biology approaches elucidate DnaK's role in bacterial stress response networks?

Systems biology approaches offer powerful frameworks for understanding DnaK's position within bacterial stress response networks:

• Multi-omics integration: Combine transcriptomics, proteomics, and metabolomics data from DnaK-depleted or overexpressing strains to map the systemic effects of perturbing DnaK function.

• Protein-protein interaction networks: Use techniques like affinity purification-mass spectrometry to comprehensively map DnaK's interactome under different stress conditions. The search results indicate DnaK "associated with many essential clients" .

• Genetic interaction mapping: Perform synthetic genetic array analysis with DnaK mutants to identify genes that buffer or exacerbate DnaK perturbation.

• Temporal profiling: Track dynamic changes in DnaK interactions and cellular responses following stress induction to develop time-resolved models of stress response networks.

• Mathematical modeling: Develop computational models incorporating DnaK's ATPase cycle, co-chaperone interactions, and client binding to predict system-level behaviors.

• Network perturbation analysis: Systematically perturb components of the protein quality control network to determine how DnaK functions within redundant or compensatory pathways.

• Single-cell analyses: Examine cell-to-cell variability in DnaK expression and activity to understand how population heterogeneity contributes to stress resilience.

These approaches can elucidate how DnaK coordinates with other cellular components to maintain proteostasis under various stress conditions and potentially reveal non-canonical functions beyond its established chaperone role.