Recombinant Pelodictyon phaeoclathratiforme ATP-dependent zinc metalloprotease FtsH (ftsH)

What is Pelodictyon phaeoclathratiforme ATP-dependent zinc metalloprotease FtsH?

P. phaeoclathratiforme FtsH is a membrane-bound ATP-dependent metalloprotease with a molecular mass of approximately 70 kDa. It belongs to the AAA+ protein family (ATPases Associated with diverse cellular Activities) characterized by a conserved module of about 200 amino acid residues containing an ATP-binding site . The full-length protein consists of 662 amino acids and contains transmembrane domains that anchor it to the cytoplasmic membrane, with the majority of the protein exposed to the cytoplasm .

Methodologically, researchers identify FtsH through sequence homology with other bacterial FtsH proteins, particularly focusing on the HEXXH motif characteristic of zinc metalloproteases and the Walker A and B motifs involved in ATP binding and hydrolysis. For recombinant production, the full-length protein is typically expressed with an N-terminal His-tag in E. coli expression systems .

What is the structural organization of FtsH proteins?

FtsH proteins exhibit a hexameric structure consisting of two distinct ring formations. The protease domains form a flat hexagon with an all-helical fold, which is covered by a toroid built by the AAA domains . This architecture creates an interior chamber where proteolytic sites are located.

The active site of FtsH contains a zinc ion coordinated by two histidine residues from the conserved 423HEXXH427 motif and, contrary to earlier reports, by Asp-500 rather than Glu-486 as the third ligand . The Asp-500 residue is absolutely conserved and located at the beginning of helix α15, while Glu-486 appears to play a supporting role by forming hydrogen bonds that position the histidine side chains correctly for zinc coordination .

Research methodology to determine this structure typically involves X-ray crystallography of soluble FtsH constructs that retain functionality in caseinolytic and ATPase assays, complemented by site-directed mutagenesis to confirm the roles of specific residues.

How can researchers express and purify recombinant P. phaeoclathratiforme FtsH?

For efficient expression and purification of recombinant P. phaeoclathratiforme FtsH, researchers typically employ the following methodological approach:

• Clone the full-length ftsH gene (1-662 amino acids) into an expression vector with an N-terminal His-tag .

• Transform the construct into an E. coli expression strain optimized for membrane protein production.

• Induce protein expression under controlled temperature conditions (typically 18-25°C) to prevent inclusion body formation.

• Lyse cells and solubilize membrane fractions using appropriate detergents.

• Purify using nickel affinity chromatography followed by size exclusion chromatography.

• Store the purified protein in buffer containing glycerol (recommended 5-50%) to maintain stability during freeze-thaw cycles .

The purified protein is typically obtained as a lyophilized powder and should be reconstituted in deionized sterile water to a concentration of 0.1-1.0 mg/mL for experimental use . Repeated freezing and thawing should be avoided, with working aliquots stored at 4°C for up to one week.

What functional assays can be used to characterize P. phaeoclathratiforme FtsH activity?

Researchers can characterize the dual functions of P. phaeoclathratiforme FtsH using several complementary assays:

For proteolytic activity:

• Caseinolytic assays using fluorescently labeled casein as a substrate

• Degradation of known FtsH substrates (e.g., σ32, λ-CII) monitored by SDS-PAGE or western blotting

• Zinc-dependent proteolysis assays with and without metal chelators to confirm metalloprotease activity

For ATPase activity:

• Colorimetric assays measuring inorganic phosphate release

• Coupled enzyme assays linking ATP hydrolysis to NADH oxidation

For chaperone activity:

• Protein refolding assays using denatured alkaline phosphatase

• Membrane protein assembly monitoring through radiolabeled substrates

The interpretation of results should consider that soluble FtsH constructs lacking transmembrane domains may show uncoupled ATPase and proteolytic activities, potentially explaining their inability to degrade certain substrates like σ32 .

What is the significance of the zinc-binding site in P. phaeoclathratiforme FtsH?

The zinc-binding site in P. phaeoclathratiforme FtsH represents a critical element for its proteolytic function and classification as an Asp-zincin metalloprotease. Structural studies have revealed that the active site contains zinc coordinated by the conserved motif 423HEXXH427 and, importantly, by Asp-500 . This finding contradicts previous reports suggesting Glu-486 as the third zinc ligand.

Site-directed mutagenesis experiments demonstrate that mutation of Asp-500 to alanine completely abolishes proteolytic activity, confirming its essential role in zinc coordination . In contrast, Glu-486 appears to play a supporting role by forming hydrogen bonds to Thr-494 and to the first histidine of the HEXXH motif, explaining why Glu-486Val mutants retain approximately 10% residual proteolytic activity .

Methodologically, researchers investigating the zinc-binding site should employ:

• Site-directed mutagenesis targeting conserved residues

• Metal-dependent activity assays with zinc supplementation or chelation

• Structural studies using X-ray crystallography of both wild-type and mutant proteins

• Isothermal titration calorimetry to measure zinc binding affinity

Understanding the precise architecture of the zinc-binding site is essential for designing specific inhibitors and for elucidating the catalytic mechanism of peptide bond hydrolysis.

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

The hexameric architecture of FtsH plays a fundamental role in coupling ATP hydrolysis to protein degradation through a coordinated mechanism. The structure reveals a striking feature: the breakdown of expected hexagonal symmetry in the AAA ring, suggesting a potential requirement for symmetry mismatch between ATPase and protease moieties during the catalytic cycle .

This symmetry reduction resembles that observed in T7 gene 4 ring helicase, where distortion from C6 to C2 symmetry has been interpreted as facilitating sequential nucleotide hydrolysis and translocation . In FtsH, this asymmetry may enable a mechanism where conformational changes in the AAA domains create an effective movement or "pulling" of the target polypeptide chain toward the interior of the hexamer using conserved phenylalanine residues (e.g., Phe-234) .

Research methodologies to investigate this mechanism include:

• Cryo-electron microscopy of FtsH in different nucleotide-bound states

• FRET-based approaches to monitor conformational changes during catalysis

• Cross-linking experiments to trap substrate-bound intermediates

• Single-molecule force measurements to quantify the "pulling" force generated

The lack of coupling between ATP hydrolysis and proteolysis in truncated FtsH constructs lacking transmembrane domains might be explained by the absence of "elastic springs" formed by these transmembrane helices, which would normally restrain free movement of the AAA domains and prevent them from being locked in intermediate conformations .

What is the evolutionary significance of P. phaeoclathratiforme FtsH in photosynthetic bacteria?

Pelodictyon phaeoclathratiforme belongs to the group of green sulfur bacteria containing bacteriochlorophyll e , suggesting that its FtsH protease may have specialized functions related to photosynthetic processes. The evolutionary context of P. phaeoclathratiforme is particularly interesting as green filamentous anoxygenic phototrophic bacteria are considered among the most ancient representatives of phototrophic microorganisms .

FtsH proteases in photosynthetic organisms typically play crucial roles in:

• Turnover of photosystem components damaged by photo-oxidative stress

• Regulation of chlorophyll and bacteriochlorophyll biosynthesis

• Quality control of membrane protein complexes involved in light harvesting

Research approaches to investigate the evolutionary aspects include:

• Comparative genomic analysis of ftsH genes across photosynthetic lineages

• Phylogenetic reconstruction to trace the evolution of specialized functions

• Functional complementation studies in heterologous hosts

The study of P. phaeoclathratiforme FtsH could provide insights into the adaptation of proteolytic systems to photosynthetic lifestyles and the evolution of protein quality control mechanisms in early photosynthetic organisms.

How can researchers address the challenges in expressing functional recombinant P. phaeoclathratiforme FtsH?

Expressing functional recombinant P. phaeoclathratiforme FtsH presents several challenges due to its membrane-associated nature and complex oligomeric structure. Researchers can address these challenges through the following methodological approaches:

For truncated constructs lacking transmembrane domains, researchers should be aware that while these constructs maintain caseinolytic and ATPase activities, they may exhibit uncoupled ATP hydrolysis and proteolysis, potentially affecting their ability to degrade certain physiological substrates like σ32 .

What analytical techniques are most effective for studying P. phaeoclathratiforme FtsH structure-function relationships?

Understanding the structure-function relationships of P. phaeoclathratiforme FtsH requires a multi-technique approach combining structural, biochemical, and biophysical methods:

• Structural techniques:

  • X-ray crystallography for high-resolution static structures

  • Cryo-electron microscopy for visualizing different conformational states

  • Hydrogen-deuterium exchange mass spectrometry to map dynamic regions

  • Small-angle X-ray scattering for solution-state conformational analysis

• Functional analysis:

  • Site-directed mutagenesis coupled with activity assays to identify critical residues

  • ATPase activity measurements using malachite green or coupled enzyme assays

  • Proteolytic activity assays with model substrates and natural targets

• Interaction studies:

  • Surface plasmon resonance to measure substrate binding kinetics

  • Crosslinking mass spectrometry to identify substrate interaction sites

  • Blue native PAGE to analyze oligomeric states and complex formation

• Computational approaches:

  • Molecular dynamics simulations to model conformational changes

  • Sequence-based evolutionary analysis to identify conserved functional motifs

  • Docking studies to predict substrate recognition mechanisms

These methodologies should be selected based on the specific research question, with particular attention to maintaining the native oligomeric state and membrane environment when relevant to the biological function being investigated.

How does P. phaeoclathratiforme FtsH compare to FtsH proteins from other bacterial species?

Comparative analysis of FtsH proteins across bacterial species reveals both conserved features and specialized adaptations. The table below summarizes key characteristics of FtsH proteins from different bacterial sources:

The conserved features across all FtsH proteins include:

• Dual transmembrane anchoring regions

• AAA+ ATPase domain with conserved Walker A/B motifs

• Zinc-dependent metalloprotease domain with HEXXH motif

• Hexameric quaternary structure

Methodologically, comparative studies should employ sequence alignment tools, homology modeling, and ideally structural studies of multiple FtsH proteins to identify both conserved functional elements and species-specific adaptations.

What is the relationship between P. phaeoclathratiforme FtsH and the photosynthetic apparatus?

While direct experimental evidence specific to P. phaeoclathratiforme FtsH is limited in the search results, insights can be drawn from studies of FtsH proteins in other photosynthetic organisms. P. phaeoclathratiforme is a green sulfur bacterium containing bacteriochlorophyll e , suggesting its FtsH likely plays roles in photosynthetic protein quality control.

In photosynthetic bacteria and chloroplasts, FtsH proteases typically:

• Participate in the repair cycle of photosystems by removing damaged components

• Regulate the assembly of photosynthetic complexes

• Control the levels of bacteriochlorophyll biosynthesis enzymes

Research approaches to investigate this relationship include:

• Knockout or depletion studies to observe effects on photosynthetic efficiency

• Co-immunoprecipitation to identify interactions with photosynthetic proteins

• Proteomics analysis to determine the degradome of P. phaeoclathratiforme FtsH

• Comparative genomics with other photosynthetic bacteria to identify conserved regulatory elements

Understanding the role of FtsH in photosynthetic processes in P. phaeoclathratiforme would contribute to our knowledge of how these ancient photosynthetic bacteria maintain their photosynthetic apparatus under varying environmental conditions.

What are the most promising applications of recombinant P. phaeoclathratiforme FtsH in research?

Recombinant P. phaeoclathratiforme FtsH offers several promising research applications:

• Model system for structure-function studies:
  The availability of recombinant P. phaeoclathratiforme FtsH with His-tag provides an excellent model system for detailed structural and mechanistic investigations of this important protease family.

• Tool for studying membrane protein quality control:
  The dual protease and chaperone activities of FtsH make it valuable for investigating fundamental mechanisms of membrane protein quality control in bacteria.

• Comparative studies of ATP-dependent proteases:
  FtsH represents one class of ATP-dependent proteases, allowing comparative studies with other systems like Clp, Lon, and HslUV proteases to reveal common principles and unique features.

• Investigation of photosynthetic adaptation:
  Given the photosynthetic nature of P. phaeoclathratiforme , its FtsH could reveal specialized adaptations for protein quality control in photosynthetic membranes.

• Evolutionary studies of conserved proteolytic systems:
  As FtsH is universally conserved in bacteria with orthologs in chloroplasts and mitochondria , P. phaeoclathratiforme FtsH can serve as a model for evolutionary studies of these essential quality control systems.

Research utilizing recombinant P. phaeoclathratiforme FtsH should focus on establishing reliable functional assays and structural characterization methods that account for its complex oligomeric structure and membrane association.

What technologies are emerging for studying dynamic aspects of FtsH function?

Several emerging technologies are particularly promising for investigating the dynamic aspects of FtsH function:

• Time-resolved cryo-electron microscopy:
  This technique allows visualization of different conformational states during the ATP hydrolysis and proteolysis cycle, potentially revealing the structural basis of the proposed "pulling" mechanism .

• Single-molecule FRET:
  By labeling specific domains with fluorophores, researchers can track conformational changes in real-time at the single-molecule level, providing insights into the coordination between ATP hydrolysis and substrate translocation.

• Nanodiscs and styrene-maleic acid lipid particles (SMALPs):
  These membrane mimetics allow structural and functional studies of FtsH in a more native-like environment, potentially resolving the coupling mechanism between transmembrane domains and the AAA/protease rings.

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS):
  This technique can map conformational dynamics and substrate interactions across different functional states of the FtsH hexamer.

• In-cell structural biology:
  Emerging methods for structural studies within cells could reveal how FtsH functions in its native cellular context and how it interacts with other quality control systems.

Implementation of these technologies requires careful experimental design to maintain functional integrity while enabling high-resolution analysis of dynamic processes.