Recombinant Pseudomonas syringae pv. tomato ATP-dependent protease ATPase subunit HslU (hslU)

What is the structural and functional role of HslU in Pseudomonas syringae?

HslU is an ATP-dependent protease ATPase subunit that forms part of the HslVU protease complex. This complex plays a critical role in protein quality control and degradation pathways in prokaryotes, including P. syringae. The HslU component functions as the ATPase that provides energy for unfolding substrate proteins before their degradation by the HslV peptidase component .

Structurally, HslU forms a hexameric ring with a central pore, and its function depends on several key domains:

• N-terminal domain

• Intermediate (I) domain

• C-terminal domain containing the ATP-binding site

How does HslU interact with HslV to form a functional protease complex?

Two distinct docking modes have been observed for HslU-HslV interaction:

• X-ray mode: HslU docks to HslV with the I domains pointing toward the HslV particle. This mode would not require substrate translocation through the central pore of the HslU hexamer. Instead, HslU could deliver substrates by shuttling between high and low affinity states for HslV.

• EM mode: Electron microscopy images suggest binding of HslU to HslV with the I domains distal to HslV .

The productive complex formation between these components is essential for ATP-dependent protein degradation, but the exact mechanism remained unclear for some time due to the different docking modes observed in crystallographic versus electron microscopy studies .

What expression systems are commonly used for recombinant HslU production?

While the search results don't specifically mention expression systems for P. syringae HslU, we can draw parallels from similar recombinant protein production strategies. Common expression systems include:

• Bacterial expression (E. coli): Most commonly used for prokaryotic proteins

• Yeast expression systems

• Baculovirus-infected insect cells

• Mammalian cell expression systems

The selection of an appropriate expression system depends on research goals, required protein yields, post-translational modifications needed, and downstream applications.

What are the standard assays for measuring HslU activity?

Several established assays can be used to evaluate different aspects of HslU activity:

• ATPase activity assay: Measures the amount of inorganic phosphate formed during ATP hydrolysis, detected at 660 nm as a complex with malachite green and ammonium molybdate .

• Peptide hydrolysis assay: Uses chromogenic peptides such as carbobenzoxy-Gly-Gly-Leu-7-amido-4-methylcoumarin (Z-Gly-Gly-Leu-AMC) as substrate, requiring both HslV and HslU components .

• Protein degradation assay: Utilizes model protein substrates such as resorufin-labeled casein or FITC-labeled casein. The proteolytic activity can be measured by following manufacturer protocols or using HPLC for detection of degradation products .

How can I optimize the purification of recombinant HslU from P. syringae?

While specific purification protocols for P. syringae HslU are not detailed in the search results, a general optimized protocol would include:

• Design an expression construct with an appropriate affinity tag (His-tag, GST, etc.)

• Express in a compatible host system (typically E. coli)

• Cell lysis under conditions that maintain protein stability

• Affinity chromatography as the primary purification step

• Size exclusion chromatography to ensure homogeneity of the hexameric complex

• Quality control by SDS-PAGE, western blotting, and activity assays

ATP or non-hydrolyzable ATP analogs like AMP-PNP may be included in buffers to stabilize the hexameric structure during purification, as indicated by structural studies of the complex .

How can I evaluate HslU-HslV complex formation in vitro?

To investigate the formation and stability of the HslU-HslV complex, consider these methodological approaches:

• Size exclusion chromatography: To analyze complex formation based on molecular size changes

• Analytical ultracentrifugation: For determining complex stoichiometry and binding affinities

• Isothermal titration calorimetry (ITC): To measure thermodynamic parameters of complex formation

• Surface plasmon resonance (SPR): For real-time binding kinetics

• Electron microscopy: To visualize complex architecture and conformational states, as has been previously done to identify the EM docking mode

How do mutations in ATP-binding sites affect HslU function?

Mutational studies around the ATP-binding site have revealed several critical residues for HslU function:

Studies have shown that mutations in these key residues significantly impact the ATP hydrolysis capability of HslU and consequently affect its ability to drive protein degradation by the HslVU complex. The abundance of basic and acidic residues near the scissile anhydride bond between β- and γ-phosphates of ATP suggests that both base and acid catalysis mechanisms may be involved in the hydrolysis reaction .

What is the relationship between HslU function and type III secretion system (T3SS) in P. syringae pathogenicity?

While direct interactions between HslU and the T3SS aren't explicitly described in the search results, we can infer potential relationships based on their roles:

P. syringae utilizes the T3SS to deliver virulence-related factors called type III effectors (T3E) into plant cells. These T3E proteins promote pathogenicity or suppress host immune defenses . As a component of the protein quality control system, HslU might play an indirect role by:

• Regulating the stability and turnover of T3SS components

• Ensuring proper folding of secreted effectors

• Degrading misfolded or damaged proteins that could otherwise impair T3SS function

This relationship represents an important area for future research to determine whether HslU has specific roles in regulating virulence factor production or secretion.

How does ATP hydrolysis by HslU drive conformational changes in the protease complex?

ATP hydrolysis by HslU drives critical conformational changes necessary for substrate processing by the HslVU protease complex. Based on structural studies:

• ATP binding causes conformational changes in HslU that promote its association with HslV

• The hexameric pore of HslU is involved in substrate recognition and translocation, particularly for larger protein substrates like the maltose-binding protein-SulA fusion protein, but appears less critical for small peptide substrates and casein

• Mutations around this pore affect protein substrate processing but not small peptide hydrolysis

• The "sensor arginine" (R393) and "arginine finger" (R325) play essential roles in coordinating ATP hydrolysis with the conformational changes needed for substrate processing

The specific molecular mechanisms and conformational trajectories remain areas of active investigation.

How does P. syringae HslU compare with homologous proteins in other bacterial species?

HslU is conserved across many bacterial species, with notable homologs in:

• Other Pseudomonas species

• Campylobacter jejuni

• E. coli (the most extensively studied homolog)

While structural similarities exist, functional specificities may differ. For example, in C. jejuni, a related bacterium, HslU functions as part of the ATP-dependent protease complex and is classified as an ATP-dependent protease ATPase subunit . This protein shows sequence and functional conservation with P. syringae HslU, though species-specific adaptations likely exist.

Comparative structural and functional analyses between these homologs can provide insights into conserved mechanisms and species-specific adaptations.

What techniques can be used to investigate HslU interactions with potential substrate proteins?

To identify and characterize HslU substrate interactions, researchers can employ:

• Co-immunoprecipitation: To pull down native HslU-substrate complexes

• Yeast two-hybrid screening: For identifying potential protein interactions

• Cross-linking coupled with mass spectrometry: To capture transient interactions

• Fluorescence resonance energy transfer (FRET): For investigating dynamic interactions in real-time

• Protein microarrays: To screen for interactions with multiple potential substrates simultaneously

• Hydrogen-deuterium exchange mass spectrometry: To map interaction interfaces

For validating specific interactions, researchers often develop substrate degradation assays using purified components and fluorescently labeled model substrates such as FITC-casein .

How do different HslU domains contribute to substrate specificity?

HslU contains multiple domains that contribute to substrate recognition and processing:

• N-terminal domain: May contribute to substrate binding and recognition

• Intermediate (I) domain: Critical for interactions with HslV and potentially involved in substrate specificity

• C-terminal domain: Contains the ATP-binding site necessary for energy-dependent conformational changes

Mutational studies have shown that the HslVU activity against different substrates displays varying sensitivity to mutations in these domains. For example:

• Proteolytic activity against small peptide substrates and casein is relatively robust to mutations

• Activity against more complex substrates like maltose-binding protein-SulA fusion protein depends on the presence of the I domain and is sensitive to mutations in both N-terminal and C-terminal domains

This suggests that different domains play specialized roles in processing various substrate types.

What are common issues in expressing and purifying active recombinant HslU?

Common challenges researchers encounter include:

• Poor solubility: HslU may form inclusion bodies, requiring refolding protocols or solubility tags

• Oligomeric state instability: The hexameric form may dissociate during purification

• ATP dependency: Proper folding and stability may require ATP or analogs during purification

• Co-purification contaminants: Host proteases may co-purify with HslU

• Activity loss during storage: Freeze-thaw cycles may disrupt the oligomeric structure

Potential solutions include:

• Adding ATP or non-hydrolyzable analogs like AMP-PNP to stabilize the structure

• Using protease inhibitors during purification

• Optimizing buffer conditions (pH, salt concentration, glycerol)

• Altering expression temperature or induction conditions

• Testing different affinity tags and their positions (N- or C-terminal)

How can I properly validate HslU-HslV complex activity in experimental systems?

A comprehensive validation approach should include multiple assays:

• Biochemical assays:

  • ATPase activity using malachite green/ammonium molybdate detection method

  • Peptidase activity using Z-Gly-Gly-Leu-AMC or similar chromogenic/fluorogenic peptides

  • Protein degradation using resorufin-labeled or FITC-labeled casein

• Controls to include:

  • ATP-binding site mutants (R393, R325) as negative controls

  • HslU or HslV alone to confirm complex dependency

  • Heat-inactivated samples

  • Non-hydrolyzable ATP analogs (AMP-PNP) to distinguish ATP-binding from hydrolysis requirements

• Activity conditions optimization:

  • Buffer composition (Tris-HCl, pH 7.5, MgCl₂)

  • Temperature and time course

  • Component ratios (e.g., 1 μg HslV with 2.5 μg HslU for peptide assays)

What are emerging approaches for studying HslU dynamics in vivo?

Cutting-edge approaches for investigating HslU dynamics within living bacterial cells include:

• CRISPR-Cas9 genome editing: For tagging endogenous HslU with fluorescent proteins or creating conditional knockouts

• Single-molecule tracking: To follow HslU movement and interactions in living cells

• Split fluorescent protein systems: For visualizing protein-protein interactions in real-time

• Transcriptomics and proteomics: To identify global effects of HslU mutation or deletion

• Cryo-electron tomography: For visualizing native HslU complexes within the cellular context

These approaches would complement the structural and biochemical studies that have dominated previous research .

How might HslU function in bacterial stress response and antibiotic resistance?

As a component of the protein quality control system, HslU likely contributes to bacterial stress responses through:

• Clearing damaged or misfolded proteins: During heat shock, oxidative stress, or antibiotic exposure

• Regulating turnover of stress-response factors: Potentially including virulence factors

• Contributing to biofilm formation: Through regulation of cell surface proteins

• Mediating antibiotic tolerance: By degrading drug-damaged proteins or drug targets

Future research might explore HslU as a potential target for antimicrobial development, especially given its essential role in protein quality control and the current understanding of its ATP-binding pocket from crystallographic studies .