Recombinant Helicobacter felis ATP-dependent zinc metalloprotease FtsH (ftsH)

What are the conserved functional motifs in H. felis FtsH and their significance?

H. felis FtsH contains several key conserved motifs essential for its function:

• The HEXXH motif (commonly H423EXXH427) in the protease domain that coordinates zinc binding

• The Walker A and Walker B motifs in the AAA domain for ATP binding and hydrolysis

• The second region of homology (SRH) in the AAA module

• A third zinc ligand, which in most FtsH proteases is an aspartic acid (Asp-500 in related species)

Mutation studies in related FtsH proteins have shown that altering the third zinc ligand (Asp-500 to alanine) completely abolishes proteolytic activity, while mutation of nearby glutamate residues (e.g., Glu-486) only reduces activity by approximately 90%, indicating the precise spatial arrangement of these residues is critical for catalysis .

What are the most effective expression systems for producing recombinant H. felis FtsH?

Based on experimental data from related studies, E. coli expression systems have proven effective for the production of recombinant H. felis FtsH. Specifically, the insertion of the H. felis ftsH gene into a versatile expression vector has been demonstrated to result in successful overexpression of the protein in E. coli . When designing expression constructs, researchers should consider:

• Using E. coli strains optimized for membrane protein expression (e.g., C41(DE3), C43(DE3))

• Incorporating affinity tags (His6, GST) for purification

• Considering the expression of soluble constructs lacking the transmembrane domains for easier purification

• Optimizing induction conditions (temperature, IPTG concentration, induction time)

The transmembrane domains of FtsH have been reported to be essential for oligomerization and activity in E. coli FtsH , but soluble constructs containing only the ATPase and protease domains have been successfully crystallized and shown to retain functionality in caseinolytic and ATPase assays .

What purification strategies yield the highest purity and activity of recombinant H. felis FtsH?

For optimal purification of recombinant H. felis FtsH, a multi-step approach is recommended:

• Membrane Fraction Isolation: If expressing the full-length protein with transmembrane domains, isolate membrane fractions using ultracentrifugation.

• Detergent Solubilization: Solubilize the membrane fraction using mild detergents like n-dodecyl-β-D-maltoside (DDM) or CHAPS.

• Affinity Chromatography: Use immobilized metal affinity chromatography (IMAC) for His-tagged proteins or glutathione affinity for GST-tagged constructs.

• Ion Exchange Chromatography: Apply the eluate to an ion exchange column for further purification.

• Size Exclusion Chromatography: Perform gel filtration to isolate the hexameric form and remove aggregates.

For soluble constructs lacking transmembrane domains, similar approaches to those used for H. pylori FtsH ATPase domain purification can be employed . Activity assays at each purification step can help track the retention of enzymatic function.

How can the proteolytic activity of recombinant H. felis FtsH be measured?

The proteolytic activity of recombinant H. felis FtsH can be assessed using several established methods:

• Caseinolytic Assays: Using fluorescently labeled casein as a substrate and measuring the increase in fluorescence upon proteolysis .

• Specific Substrate Degradation: Monitoring the degradation of known FtsH substrates such as σ32, λcII, or SecY using SDS-PAGE and western blotting .

• FRET-based Assays: Employing peptide substrates with fluorescence resonance energy transfer (FRET) pairs that change signal upon cleavage.

When measuring proteolytic activity, it's crucial to include appropriate controls:

• Negative control: FtsH with mutations in the catalytic site (e.g., D500A mutation)

• Positive control: Well-characterized FtsH from E. coli

• ATP-dependency control: Assays conducted with and without ATP

The expected ATP consumption for FtsH activity is approximately 8 ATP molecules per peptide cleavage based on experimental data, with a theoretical minimum of 6 ATP molecules per cleavage event .

What methods can be used to study the ATPase activity of H. felis FtsH?

The ATPase activity of H. felis FtsH can be investigated using these methodologies:

• Colorimetric Phosphate Detection: Using malachite green or other reagents to detect inorganic phosphate released during ATP hydrolysis.

• Coupled Enzyme Assays: Linking ATP hydrolysis to NADH oxidation through pyruvate kinase and lactate dehydrogenase, monitoring the decrease in NADH absorbance at 340 nm.

• Radioactive ATP Assays: Using [γ-32P]ATP and measuring the release of 32P-labeled inorganic phosphate.

For accurate assessment of ATPase activity:

• Conduct assays at physiologically relevant temperatures (37°C for H. felis)

• Include appropriate metal cofactors (typically Mg2+)

• Assess the effect of substrate presence on ATPase activity, as substrate binding may stimulate ATP hydrolysis

The ATPase activity of FtsH is essential for its proteolytic function, as it provides the energy required for substrate unfolding and translocation through the central pore to the proteolytic site .

How can researchers investigate the substrate specificity of H. felis FtsH?

To investigate the substrate specificity of H. felis FtsH, researchers can employ these approaches:

• Candidate Substrate Testing: Express and purify potential substrate proteins (based on known FtsH substrates from other bacteria) and assess their degradation by H. felis FtsH in vitro.

• Proteomics Approaches: Compare the proteome of H. felis strains with functional versus inactive FtsH to identify accumulated substrates.

• Site-Directed Mutagenesis: Modify the substrate-binding regions of FtsH (e.g., the phenylalanine residue in the FVG motif near the central pore of the ATPase domain) to alter substrate recognition.

• Chimeric FtsH Constructs: Create chimeric proteins with domains from H. felis FtsH and other FtsH proteins to map substrate specificity determinants.

When analyzing substrate specificity, consider both membrane and cytosolic substrates, as FtsH is known to degrade both misassembled membrane proteins (SecY, subunit a of FoF1-ATPase, YccA) and short-lived soluble regulatory proteins (σ32, LpxC, λcII) .

What are the recommended approaches for crystallization of H. felis FtsH for X-ray crystallography?

For successful crystallization of H. felis FtsH, researchers should consider these strategies based on previous successes with related FtsH proteins:

• Construct Design:

  • Create truncated constructs excluding the transmembrane domains, focusing on the cytosolic region containing the ATPase and protease domains

  • Consider crystallizing individual domains separately, as has been done for the H. pylori FtsH ATPase domain

• Protein Preparation:

  • Ensure high protein purity (>95%) and monodispersity by size exclusion chromatography

  • Verify protein folding using circular dichroism or thermal shift assays

  • Test protein stability with and without nucleotides (ATP/ADP) and zinc

• Crystallization Conditions:

  • Screen with commercial sparse matrix screens optimized for soluble proteins

  • For complexes with ADP or non-hydrolyzable ATP analogs, include these ligands during crystallization

  • Consider the addition of zinc or other metal ions that might stabilize the protease domain

• Crystal Optimization:

  • Use seeding techniques to improve crystal quality

  • Test cryoprotectants carefully to avoid damaging delicate crystals

Based on previous FtsH structures, researchers might expect hexameric arrangements with potential asymmetry between protease and ATPase rings .

How can researchers investigate the oligomeric state and conformational changes of H. felis FtsH?

To investigate the oligomeric state and conformational dynamics of H. felis FtsH, several biophysical techniques can be employed:

• Size Exclusion Chromatography with Multi-Angle Light Scattering (SEC-MALS):

  • Determines absolute molecular weight in solution

  • Can confirm the hexameric assembly (expected ~420 kDa for full-length hexamer)

• Analytical Ultracentrifugation (AUC):

  • Provides information on oligomeric state and shape

  • Can detect multiple oligomeric forms in equilibrium

• Negative Stain Electron Microscopy:

  • Visualizes the ring-shaped toroidal oligomers typically formed by FtsH

  • Can observe conformational heterogeneity in the sample

• Cryo-Electron Microscopy:

  • For high-resolution structural studies of the full-length protein in different nucleotide states

  • Can potentially capture different conformational states during the catalytic cycle

• Small-Angle X-ray Scattering (SAXS):

  • Provides low-resolution structural information in solution

  • Can detect large conformational changes upon nucleotide binding

Studies on related FtsH proteins have revealed conformational changes between nucleotide-free and ADP-bound states, particularly in the H. pylori FtsH ATPase domain, suggesting mechanical forces for substrate translocation . Similar conformational dynamics might be expected for H. felis FtsH.

What is the relationship between H. felis FtsH activity and co-infection with H. pylori?

The relationship between H. felis FtsH activity and co-infection with H. pylori represents an important area for investigation based on clinical findings:

• Co-infection Prevalence: Clinical studies have shown that H. felis is often found in patients already infected with H. pylori. In one study, PCR detection found H. felis in 4% of patients, and all these patients were also positive for H. pylori .

• Pathological Correlations: Co-infection of H. felis with H. pylori appeared to be associated with increased rates of intestinal metaplasia (IM):

  • IM was observed in 20% of patients with H. felis and H. pylori co-infection

  • IM was observed in only 3% of patients with H. pylori infection alone

This data suggests potential synergistic pathological effects during co-infection, as shown in the following table from clinical studies:

While this sample size is too small for statistical significance, it suggests a trend that warrants further investigation .

The specific role of H. felis FtsH in these co-infection scenarios remains to be elucidated, but possible mechanisms include:

• Altered stress responses in the presence of both bacterial species

• Changes in membrane protein composition affecting host-pathogen interactions

• Modified proteolytic processing of virulence factors

What phylogenetic relationships exist among FtsH proteins from different Helicobacter species?

Phylogenetic analysis of FtsH proteins from various Helicobacter species reveals evolutionary relationships that can inform functional studies. While comprehensive phylogenetic data specific to Helicobacter FtsH proteins is limited in the provided search results, general patterns can be inferred:

• Sequence Conservation: FtsH is universally conserved in bacteria, with high sequence similarity in the ATPase and protease domains across different bacterial species .

• Helicobacter-Specific Features: Sequencing data indicates that H. felis FtsH shares significant sequence similarity with FtsH proteins from other Helicobacter species, reflecting their common ancestry.

• Functional Domains: The core functional elements—including the HEXXH motif in the protease domain and the Walker A/B motifs in the ATPase domain—are highly conserved across Helicobacter species.

When H. felis DNA sequences were analyzed, PCR product sequences of the ureaseB gene of H. heilmannii had 100% similarity to 'Candidatus H. heilmannii strains' (GenBank: AF508012 and L25079), while H. felis sequences had 100% similarity to strains GenBank: FQ670179 and X69080 . This indicates clear phylogenetic distinction between Helicobacter species despite functional conservation of key proteins like FtsH.

How can recombinant H. felis FtsH be utilized as a tool for studying bacterial membrane protein quality control?

Recombinant H. felis FtsH offers valuable opportunities for studying bacterial membrane protein quality control mechanisms:

• In Vitro Reconstitution Systems:

  • Purified recombinant H. felis FtsH can be incorporated into liposomes or nanodiscs to create minimal membrane protein quality control systems

  • These systems allow controlled study of substrate recognition, unfolding, and degradation in defined lipid environments

• Substrate Profiling:

  • Systematic testing of membrane protein substrates can reveal recognition motifs and degradation signals

  • Comparison with FtsH from other bacteria can identify species-specific adaptations in quality control mechanisms

• Regulatory Network Mapping:

  • Using H. felis FtsH variants with mutations in key functional domains to disrupt specific aspects of its activity

  • Tracking consequent changes in membrane protein composition and cellular stress responses

• Co-factor and Regulator Identification:

  • Pull-down assays with recombinant H. felis FtsH to identify interacting proteins

  • Analysis of potential regulatory proteins similar to the HflK/C complex in E. coli that forms a "hat-like" structure containing up to four FtsH complexes and regulates FtsH activity

The transmembrane domains of FtsH have been reported to be essential for oligomerization and activity in E. coli , making studies of the full-length protein particularly valuable for understanding membrane-associated quality control mechanisms.

What techniques can be used to identify novel substrates of H. felis FtsH in vivo?

Identifying novel substrates of H. felis FtsH in vivo requires sophisticated approaches combining genetics, proteomics, and biochemistry:

• Comparative Proteomics:

  • Compare protein abundance in wild-type H. felis versus FtsH-deficient or catalytically inactive FtsH mutant strains

  • Proteins that accumulate in FtsH-deficient strains are potential substrates

  • Use stable isotope labeling (SILAC) or isobaric tagging (TMT/iTRAQ) for quantitative comparison

• Protein Stability Profiling:

  • Pulse-chase experiments with radiolabeled amino acids or non-radioactive analogs

  • Compare protein half-lives in wild-type versus FtsH-deficient strains

  • Proteins with extended half-lives in FtsH-deficient strains are candidate substrates

• Substrate Trapping:

  • Generate catalytically inactive "trap" variants of H. felis FtsH that bind but cannot degrade substrates

  • Use affinity purification followed by mass spectrometry to identify trapped proteins

  • Verify with in vitro degradation assays using purified components

• In Vivo Crosslinking:

  • Incorporate photo-activatable amino acids into FtsH using expanded genetic code techniques

  • UV-induce crosslinking followed by purification and mass spectrometry

  • Identifies transient interactions with potential substrates

These approaches can reveal both membrane and soluble substrates, as FtsH is known to degrade both misassembled membrane proteins and short-lived soluble regulatory proteins .

What are common challenges in expressing and purifying active recombinant H. felis FtsH and how can they be addressed?

Researchers often encounter several challenges when working with recombinant H. felis FtsH. Here are the most common issues and their solutions:

• Low Expression Levels:

  • Challenge: Membrane proteins like full-length FtsH often express poorly in heterologous systems.

  • Solutions:

    • Use specialized E. coli strains (C41/C43) designed for membrane protein expression

    • Optimize codon usage for the expression host

    • Consider fusion tags that enhance solubility (MBP, SUMO)

    • Lower induction temperature (16-20°C) and extend expression time

• Protein Aggregation:

  • Challenge: FtsH tends to form inclusion bodies or aggregates when overexpressed.

  • Solutions:

    • Express at lower temperatures with reduced inducer concentration

    • Include stabilizing agents (glycerol, specific detergents) in lysis buffers

    • Consider expressing soluble domains separately if full-length protein is problematic

    • Optimize detergent type and concentration for membrane extraction

• Loss of Zinc During Purification:

  • Challenge: The catalytic zinc ion can be lost during purification, reducing activity.

  • Solutions:

    • Include low concentrations of zinc (1-10 μM ZnCl₂) in purification buffers

    • Avoid strong chelating agents like EDTA

    • Verify zinc content using colorimetric assays or atomic absorption spectroscopy

• Oligomerization Issues:

  • Challenge: Obtaining homogeneous hexameric assemblies can be difficult.

  • Solutions:

    • Use size exclusion chromatography as a final purification step

    • Include ATP or non-hydrolyzable analogs to stabilize the hexameric form

    • Optimize detergent:protein ratio to prevent artificial aggregation

• Low Enzymatic Activity:

  • Challenge: Purified protein may show limited proteolytic activity.

  • Solutions:

    • Ensure presence of both zinc and magnesium in activity assays

    • Verify ATP hydrolysis is occurring (prerequisite for proteolytic activity)

    • Test activity against known FtsH substrates from related bacteria

    • Optimize buffer conditions (pH, salt concentration) for maximal activity

How can researchers resolve inconsistencies in experimental results when studying H. felis FtsH function?

When faced with inconsistent results in H. felis FtsH functional studies, researchers should consider these methodical troubleshooting approaches:

• Protein Quality Assessment:

  • Issue: Variation in protein preparation quality can cause inconsistent results.

  • Resolution:

    • Implement rigorous quality control checks: SDS-PAGE, SEC-MALS, thermal stability assays

    • Verify oligomeric state before each experiment

    • Test ATPase activity as a proxy for proper folding

    • Develop standard preparation protocols with specific acceptance criteria

• Assay Condition Optimization:

  • Issue: FtsH activity is sensitive to buffer conditions, potentially leading to variable results.

  • Resolution:

    • Systematic testing of buffer components (pH, salt, metal ions)

    • Control temperature precisely during assays

    • Standardize ATP:Mg²⁺ ratios

    • Include appropriate positive and negative controls in each experiment

• Substrate Variation:

  • Issue: Different substrate preparations may have variable susceptibility to degradation.

  • Resolution:

    • Use well-characterized, homogeneous substrate preparations

    • Consider the folding state of the substrate (some require partial unfolding)

    • Verify substrate quality prior to degradation assays

    • Test multiple substrate concentrations to determine optimal enzyme:substrate ratios

• Experimental Design Considerations:

  • Issue: Complex experimental designs can introduce variables affecting reproducibility.

  • Resolution:

    • Implement factorial experimental designs to identify interacting variables

    • Use internal standards where possible

    • Develop quantitative readouts rather than qualitative assessments

    • Consider time-course experiments rather than single timepoint measurements

• Data Analysis Approaches:

  • Issue: Different analysis methods can lead to different interpretations of the same data.

  • Resolution:

    • Establish standard data analysis workflows

    • Use multiple analytical approaches to confirm findings

    • Implement appropriate statistical tests for significance

    • Consider Bayesian approaches for handling variable data

By methodically addressing these potential sources of inconsistency, researchers can develop more robust and reproducible protocols for studying H. felis FtsH function.

What are the most promising research avenues for understanding H. felis FtsH in gastric disease models?

Several promising research directions could advance our understanding of H. felis FtsH in gastric disease models:

• Genetic Manipulation Studies:

  • Generate H. felis strains with modified FtsH (point mutations, domain deletions)

  • Assess colonization efficiency and pathogenicity in mouse models

  • Determine if FtsH activity correlates with disease severity

• Host-Pathogen Interaction Studies:

  • Investigate how H. felis FtsH affects interaction with host immune cells

  • Determine if FtsH proteolytically processes bacterial surface proteins involved in adhesion

  • Study the impact of FtsH on H. felis survival under host-imposed stress conditions

• Co-infection Models:

  • Develop sophisticated co-infection models with H. pylori and H. felis

  • Assess how H. felis FtsH activity influences co-infection dynamics

  • Investigate potential synergistic effects on pathology development, particularly intestinal metaplasia which showed a trend toward increased prevalence in co-infected patients

• Comparative Virulence Studies:

  • Compare colonization and virulence of wild-type H. felis versus FtsH-deficient strains

  • Assess the long-term consequences of infection (gastritis, metaplasia development)

  • Determine if phospholipase A2 expression in hosts differentially affects FtsH-deficient strains

• Therapeutic Targeting:

  • Develop specific inhibitors of H. felis FtsH

  • Test their efficacy in reducing bacterial colonization in animal models

  • Assess potential for species-specific inhibitors that target H. felis but not beneficial bacteria

These research directions could significantly advance our understanding of how this ATP-dependent protease contributes to Helicobacter pathogenesis and potentially identify new therapeutic targets.

How might structural biology approaches advance our understanding of H. felis FtsH mechanism and regulation?

Advanced structural biology approaches offer significant potential for understanding H. felis FtsH mechanisms and regulation:

• Cryo-Electron Microscopy (Cryo-EM) Studies:

  • Capture different conformational states during the ATP hydrolysis and proteolysis cycle

  • Visualize substrate engagement and translocation through the central pore

  • Potentially reveal species-specific features of H. felis FtsH compared to better-studied homologs

  • Enable visualization of regulatory protein interactions in situ

• Integrative Structural Approaches:

  • Combine X-ray crystallography of individual domains with cryo-EM of full complexes

  • Use crosslinking mass spectrometry to map domain interactions

  • Apply hydrogen-deuterium exchange mass spectrometry to identify dynamic regions

  • Develop computational models of substrate processing based on structural constraints

• In-Membrane Structural Studies:

  • Investigate FtsH structure in native-like membrane environments using:

    • Lipid nanodiscs for cryo-EM studies

    • Solid-state NMR for studying membrane-embedded regions

    • Electron crystallography of 2D crystals in lipid bilayers

• Dynamic Structural Analysis:

  • Apply single-molecule FRET to monitor conformational changes during substrate processing

  • Use time-resolved structural methods to capture transient states

  • Develop reporter constructs to monitor structural changes in vivo

• Regulatory Complex Structures:

  • Investigate potential interactions with regulatory proteins similar to the E. coli HflK/C complex

  • Determine if H. felis FtsH forms supercomplexes with other membrane proteins

  • Study the structural basis of selective substrate recognition