Recombinant Borrelia burgdorferi ATP-dependent zinc metalloprotease FtsH (ftsH)

What is Borrelia burgdorferi ATP-dependent zinc metalloprotease FtsH?

The FtsH homologous protein BB0789 in Borrelia burgdorferi is an ATP-dependent protease that plays an essential role in degrading misfolded or unneeded membrane and cytosolic proteins. This protease belongs to the AAA+ (ATPases Associated with various cellular Activities) family and is conserved across bacteria, mitochondria, and chloroplasts. In B. burgdorferi specifically, BB0789 has been demonstrated to be crucial for the spirochete's ability to survive and cause Lyme disease .

What is the structural organization of B. burgdorferi FtsH?

The crystal structure of cytosolic BB0789 (amino acids 166-614) reveals a complex arrangement consisting of two main domains: an AAA+ ATPase domain and a zinc-dependent metalloprotease domain. These domains are organized in a functionally essential hexamer ring structure. The AAA+ domain has been observed in an ADP-bound state, while the protease domain coordinates a zinc ion using two histidine residues and one aspartic acid residue. The complete hexameric structure includes a central pore region that was poorly defined in crystal studies but has been predicted using AlphaFold to provide a complete structural picture .

How does FtsH contribute to B. burgdorferi pathogenesis?

FtsH (BB0789) is absolutely necessary for B. burgdorferi to survive and cause Lyme disease. This requirement is linked to the complex life cycle of B. burgdorferi, which involves residence in both mammals and ticks. This dual-host lifestyle demands a wide range of membrane proteins and short-lived cytosolic regulatory proteins for host invasion and persistence. FtsH likely plays a critical role in protein quality control and regulation during these host adaptation processes .

What are the considerations for expressing recombinant B. burgdorferi FtsH?

When expressing recombinant B. burgdorferi FtsH, researchers should consider that the full-length protein contains N-terminal transmembrane α-helices and a small periplasmic domain. For crystallography studies, a truncated version (amino acids 166-614) lacking these regions has been successfully used. Expression systems should account for the protein's hexameric structure, which is essential for both ATPase and proteolytic activity. Additionally, the expression system must ensure proper coordination of the zinc ion in the metalloprotease domain by two histidine residues and one aspartic acid residue .

What methods can be used to assess the dual activity of FtsH?

FtsH possesses both protease and ATPase activities that can be assessed using distinct biochemical assays:

• ATPase activity assessment: Standard ATPase assays measuring ATP hydrolysis rates using methods such as colorimetric phosphate detection or coupled enzyme assays.

• Protease activity assessment: Using fluorogenic peptide substrates or monitoring the degradation of known protein substrates through techniques such as SDS-PAGE and western blotting.

These assays should be optimized considering that both activities are dependent on the hexameric structure of the protein. Researchers should ensure proper oligomerization of recombinant FtsH before activity testing .

How can zebrafish models be adapted for studying FtsH function in B. burgdorferi?

Zebrafish (Danio rerio) offer a promising high-throughput in vivo model for studying B. burgdorferi infection. To adapt this model for FtsH studies specifically:

• Utilize the established immersion infection protocol with 1×10^7 spirochetes/mL in a 1:1 mixture of BSK-H media and zebrafish water to maintain spirochete morphology and zebrafish health.

• After infection, standard molecular techniques can be employed to study FtsH function:

  • PCR and RT-PCR to detect B. burgdorferi and measure ftsH gene expression

  • Immunohistochemistry with FtsH-specific antibodies to localize the protein

  • Fluorescent microscopy to visualize infection in tissues where FtsH function might be critical

This approach would allow for in vivo assessment of FtsH function, particularly when comparing wild-type and FtsH-mutant strains of B. burgdorferi .

How does the hexameric arrangement of FtsH contribute to its function?

The hexameric ring structure of FtsH is critical for both its ATPase and proteolytic activities. The arrangement creates a central pore through which substrate proteins likely pass during processing. The hexamer forms a molecular machine where:

• The AAA+ domains use ATP hydrolysis to drive conformational changes that unfold substrate proteins and translocate them through the central pore.

• The metalloprotease domains positioned at one end of the hexamer contain active sites with zinc ions coordinated by two histidine residues and one aspartic acid residue, which cleave the unfolded substrates.

This structural arrangement allows for the coupling of ATP hydrolysis to protein unfolding and subsequent degradation, creating an efficient proteolytic system. Mutations disrupting hexamer formation would likely render the protein non-functional .

What are the specific metal coordination features of B. burgdorferi FtsH?

The metalloprotease domain of B. burgdorferi FtsH (BB0789) coordinates a zinc ion through a specific arrangement of amino acid residues. The crystal structure reveals that two histidine residues and one aspartic acid residue participate in this coordination. This metal-binding site forms the catalytic center of the protease domain.

The specific coordination geometry and the identity of any additional coordinating molecules (such as water) would be important considerations for researchers studying catalytic mechanisms or designing inhibitors. The zinc coordination is essential for the proteolytic activity of FtsH and represents a potential target for antimicrobial development .

What are the challenges in studying membrane-associated aspects of FtsH?

Studying the membrane-associated functions of FtsH presents several challenges:

• Structural complexity: The full-length FtsH contains N-terminal transmembrane α-helices and a small periplasmic domain that were excluded from the crystallized construct (166-614). These regions likely play important roles in substrate recognition and membrane interaction.

• Reconstitution systems: Developing lipid reconstitution systems that accurately mimic the B. burgdorferi membrane environment is challenging but necessary for studying how FtsH integrates into and functions within the membrane.

• Substrate identification: Identifying membrane protein substrates that are processed by FtsH in B. burgdorferi requires specialized proteomics approaches that can detect changes in the membrane proteome.

Researchers should consider combining structural biology with advanced microscopy techniques, such as cryo-electron microscopy, to visualize the full-length protein in a membrane environment .

How can computational approaches enhance our understanding of FtsH dynamics?

Computational approaches offer powerful tools for understanding the complex dynamics of FtsH:

• Molecular dynamics simulations: These can model the conformational changes that occur during ATP hydrolysis and substrate processing, providing insights into how energy from ATP hydrolysis is converted into mechanical force.

• AlphaFold and related tools: As demonstrated in the BB0789 structure study, AlphaFold can complement experimental structural data by predicting regions that are poorly defined in crystal structures, such as the loop region forming the central pore.

• Substrate docking and processing simulations: Computational docking studies can predict how potential substrates interact with the FtsH hexamer and how they might be processed through the central pore.

These approaches can guide experimental design and provide mechanistic hypotheses that can be tested through directed mutagenesis or other experimental approaches .

How does FtsH function vary between tick and mammalian host environments?

The function of FtsH in B. burgdorferi likely adapts to the dramatically different environments of tick and mammalian hosts. Key considerations include:

• Temperature adaptation: Ticks typically have lower body temperatures than mammals, which may affect the enzymatic activity and substrate specificity of FtsH.

• Nutrient availability: The different nutrient profiles in tick versus mammalian environments may influence which proteins need to be degraded by FtsH for recycling amino acids.

• Host-specific protein regulation: FtsH may selectively degrade certain B. burgdorferi proteins that are no longer needed when transitioning between hosts.

Research approaches should include comparative studies of FtsH activity and substrate profiles under conditions mimicking tick versus mammalian environments. Differential expression analysis of the ftsH gene during these transitions could also provide valuable insights .

What methods can be used to identify the in vivo substrates of FtsH in B. burgdorferi?

Identifying the in vivo substrates of FtsH is crucial for understanding its role in B. burgdorferi pathogenesis. Several approaches can be employed:

• Comparative proteomics: Compare protein profiles between wild-type B. burgdorferi and strains with conditional FtsH depletion to identify proteins that accumulate when FtsH activity is reduced.

• Proximity labeling: Utilize techniques like BioID or APEX to identify proteins that physically interact with FtsH in living bacteria.

• Stable isotope labeling: Use pulse-chase experiments with isotope-labeled amino acids to track protein degradation rates dependent on FtsH activity.

• In vivo crosslinking: Capture transient enzyme-substrate interactions through crosslinking followed by mass spectrometry.

These approaches should be performed under different conditions that mimic the tick vector and mammalian host environments to identify context-specific substrates .

What are the critical controls for assessing recombinant FtsH activity?

When assessing the activity of recombinant B. burgdorferi FtsH, several critical controls should be included:

• Negative controls:

  • Heat-inactivated FtsH to confirm the enzymatic nature of observed activity

  • Site-directed mutants with alterations to key catalytic residues (zinc-coordinating histidines and aspartic acid)

  • ATP-binding site mutants to differentiate between ATP-dependent and independent functions

• Positive controls:

  • Known FtsH substrates from related organisms

  • Confirmation of proper hexameric assembly using size exclusion chromatography or native PAGE

• Specificity controls:

  • Testing activity with protease inhibitors specific for different classes of proteases

  • Metal chelators to confirm zinc-dependence

  • Alternative nucleotides to confirm ATP specificity

These controls ensure that the observed activity is specific to properly folded and assembled FtsH rather than contaminants or non-specific effects .

How can researchers effectively model FtsH function in laboratory settings given B. burgdorferi's complex life cycle?

Modeling FtsH function across B. burgdorferi's complex life cycle requires a multi-faceted approach:

• In vitro culture conditions: Develop media formulations that mimic aspects of both tick and mammalian environments, with appropriate temperature shifts (33°C for mammalian, lower for tick).

• Animal models: The zebrafish model offers a promising approach as it allows for high-throughput in vivo studies of B. burgdorferi infection. The established immersion protocol with 1×10^7 spirochetes/mL maintains good survival rates while allowing for infection.

• Conditional expression systems: Develop strains with inducible FtsH expression to study the immediate effects of FtsH depletion or overexpression at different stages of the life cycle.

• Tissue-specific analyses: Use techniques like immunohistochemistry to localize both B. burgdorferi and FtsH activity in specific tissues, as studies have shown that spirochetes can be found in diverse tissues including eyes, gills, heart, liver, and digestive tract in zebrafish models .