Recombinant Petrotoga mobilis ATP-dependent zinc metalloprotease FtsH 3 (ftsH3)

What is the molecular structure of recombinant Petrotoga mobilis FtsH3?

Recombinant Petrotoga mobilis FtsH3 is characterized as an ATP-dependent integral membrane protease with a molecular architecture consisting of two rings. The protease domains possess an all-helical fold and form a flat hexagon that is covered by a toroid built by the AAA domains . The active site of the protease classifies FtsH as an Asp-zincin, containing an aspartic acid as the third zinc ligand, contrary to previous reports . This hexameric structure exhibits a notable breakdown of the expected hexagonal symmetry in the AAA ring, which suggests a potential requirement for symmetry mismatch between ATPase and protease moieties during the catalytic cycle .

What functional domains are present in FtsH3?

FtsH3 contains two primary functional domains: an ATPase (AAA) domain and a proteolytic domain. The AAA domain is responsible for ATP hydrolysis, which provides energy for substrate unfolding and translocation into the proteolytic chamber . The proteolytic domain contains a zinc-binding site characteristic of metalloproteases, with the active site presenting an aspartic acid as the third zinc ligand . In addition, FtsH3 possesses transmembrane domains that anchor it to the membrane, with the catalytic domains facing the matrix or cytoplasmic side depending on its cellular location .

How does FtsH3 participate in protein quality control mechanisms?

FtsH3 plays a crucial role in protein quality control by degrading unneeded or damaged membrane proteins and also targeting soluble signaling factors . Specifically in plant mitochondria, FTSH3 is involved in the disassembly of the matrix arm domain of Complex I (CI) via direct protein-protein interaction with the PSST subunit located at the interface of the membrane and matrix module . This interaction facilitates the selective degradation and turnover of damaged and non-functional subunits . Compared to other AAA+ proteases, FTSH homologs exhibit relatively weak protein unfolding activity, as measured by their efficiency in converting ATP hydrolysis to protein degradation .

What is the mechanistic basis for FtsH3's substrate recognition and specificity?

FtsH3 exhibits specific substrate recognition through direct protein-protein interactions. In Arabidopsis mitochondria, FTSH3 recognizes the PSST subunit of Complex I through a specific interaction that can be abolished by mutations in either protein . Yeast two-hybrid (Y2H) interaction assays using a proteolytically inactive form of FTSH3 (FTSH3 TRAP) demonstrated that FTSH3 directly interacts with PSST, but this interaction is lost when specific mutations are introduced (P415L in FTSH3 or S70F in PSST) .

The substrate recognition appears to depend primarily on the ATPase domain rather than the proteolytic domain, as complementation studies with proteolytically inactive FTSH3 still restored function . This suggests that the ATPase activity of FtsH3 plays a critical role in substrate recognition and the initial steps of protein degradation. FtsH proteases generally recognize substrates when they are misfolded or disassembled from their complex, allowing them to be unfolded and degraded .

How do the oligomeric states of FtsH3 influence its enzymatic activity?

FtsH3 forms complex oligomeric structures that directly impact its function. The protein can form both homo-oligomeric and hetero-oligomeric complexes . In Arabidopsis, AtFtsH3 can form hetero-oligomeric complexes with AtFtsH10 and also with prohibitins . Interestingly, when AtFtsH3 is absent (in ftsh3 mutants), the level of AtFtsH10 increases, suggesting that functions of the homo- and hetero-oligomeric complexes containing AtFtsH3 can be at least partially substituted by AtFtsH10 homo-oligomers .

The different symmetries observed between the protease and AAA rings in the hexameric structure suggest a translocation mechanism of the target polypeptide chain into the interior of the molecule where the proteolytic sites are located . The breakdown of hexagonal symmetry in the AAA ring has been interpreted in terms of sequential nucleotide hydrolysis and substrate translocation, similar to what has been observed in other ring-shaped molecular machines .

What structural elements control FtsH3's ATP-dependent proteolysis?

The ATP-dependent proteolytic activity of FtsH3 involves a sophisticated mechanism where ATP hydrolysis drives conformational changes that facilitate substrate translocation and degradation. The crystal structure reveals that the rearrangement of the AAA domains would lead to an effective movement or "pulling" of the target polypeptide chain attached to hydrophobic side chains (such as Phe-234) and move the target toward the interior of the hexamer .

This mechanism functions even when hydrolysis or ADP-ATP exchange does not occur in all six AAA domains simultaneously but rather in a probabilistic manner, as long as there is space for domain movement . The coupling between ATP hydrolysis and proteolysis may require "elastic springs" formed by the transmembrane helices that would restrain the free movement of the AAA domains and prevent them from being locked into one intermediate conformation on the reaction pathway .

What are the optimal conditions for expressing and purifying recombinant Petrotoga mobilis FtsH3?

For optimal expression and purification of recombinant Petrotoga mobilis FtsH3, researchers should consider the thermophilic nature of the source organism. Expression systems should be designed with temperature-appropriate promoters and host strains capable of proper protein folding at elevated temperatures.

Based on general protocols for membrane-bound AAA+ proteases:

• Expression system selection: E. coli BL21(DE3) or Rosetta strains are commonly used, with expression vectors containing T7 promoters.

• Temperature considerations: Since Petrotoga mobilis is thermophilic, expression at temperatures between 30-37°C may improve folding, with potential heat shock steps to improve solubility.

• Solubilization strategy: As FtsH3 is a membrane protein, detergent solubilization is critical. Commonly used detergents include n-dodecyl-β-D-maltoside (DDM), Triton X-100, or CHAPS at concentrations above their critical micelle concentration.

• Purification approach: Metal affinity chromatography using histidine tags, followed by size exclusion chromatography to separate oligomeric forms.

• Activity preservation: Include zinc ions (typically 10-50 μM ZnCl₂) and ATP (1-5 mM) in buffers to maintain proteolytic and ATPase activity.

For functional studies, a proteolytically inactive variant (FTSH3 TRAP) can be generated by site-directed mutagenesis of the active site residues in the proteolytic domain, as has been done for homologous proteins in interaction studies .

How can the interaction between FtsH3 and substrate proteins be experimentally verified?

Multiple complementary approaches can be employed to verify FtsH3-substrate interactions:

• Yeast Two-Hybrid (Y2H) assays: As demonstrated with FTSH3 and PSST, Y2H can confirm direct protein-protein interactions . For FtsH3, a proteolytically inactive form (FTSH3 TRAP) should be used to prevent degradation of interacting partners. The interaction can be detected by growth on selective media (e.g., quadruple dropout medium) .

• Co-immunoprecipitation (Co-IP): Using antibodies against FtsH3 or epitope-tagged FtsH3 to pull down interacting partners from cellular extracts, followed by mass spectrometry identification.

• Pull-down assays: Utilizing recombinant His-tagged FtsH3 immobilized on Ni-NTA resin to capture interacting proteins from cell lysates.

• Surface Plasmon Resonance (SPR): For quantitative measurement of binding kinetics between purified FtsH3 and potential substrates.

• Crosslinking coupled with mass spectrometry: Chemical crosslinking of FtsH3 with its substrates followed by mass spectrometry analysis to identify interaction sites.

• Genetic approaches: Identifying suppressor mutations (as in the rmb1 and rmb2 mutations) that disrupt the interaction and alter phenotypes related to FtsH3 function .

What assays can be used to measure the ATPase and proteolytic activities of FtsH3?

ATPase Activity Assays:

• Malachite Green Assay: Measures released inorganic phosphate from ATP hydrolysis, with sensitivity in the micromolar range.

• Coupled Enzyme Assays: Using pyruvate kinase and lactate dehydrogenase to couple ADP production to NADH oxidation, monitored spectrophotometrically at 340 nm.

• Radioactive [γ-³²P]ATP Hydrolysis: Tracking the release of radioactive phosphate for high sensitivity measurements.

Proteolytic Activity Assays:

• Fluorogenic Peptide Substrates: Monitoring the increase in fluorescence upon cleavage of quenched fluorescent peptides.

• Model Protein Degradation: Using well-characterized proteins (e.g., casein) labeled with fluorescent dyes or radioactive isotopes, and tracking their degradation over time .

• In Vitro Reconstitution Assays: With purified FtsH3 and specific substrates (such as PSST) to monitor degradation rates under different conditions .

• Proteomic Approaches: Comparative proteomics to identify accumulated substrates in FtsH3-deficient versus wild-type systems.

For studying the coupling between ATPase and proteolytic activities, researchers can examine how mutations in the ATPase domain (like P415L) affect proteolytic function, or use non-hydrolyzable ATP analogs to trap the enzyme in specific conformational states .

Comparative Properties of FtsH Proteases

Effects of Mutations on FtsH3-PSST Interaction and Function

Based on yeast two-hybrid (Y2H) interaction studies, the following mutations significantly impact FtsH3 function:

Molecular Mechanism of FtsH3-Mediated Complex I Disassembly

FtsH3 facilitates Complex I degradation through a direct interaction with the PSST subunit, located at the interface between the membrane and matrix modules of Complex I . This interaction specifically involves:

• Recognition of the PSST subunit by the ATPase domain of FtsH3

• ATP-dependent conformational changes in FtsH3 that facilitate the disassembly of the matrix arm domain

• Selective degradation of damaged or dysfunctional Complex I subunits

The process is particularly important in the context of Complex I dysfunction, as demonstrated by the restoration of Complex I abundance and activity in ciaf1 mutant plants through mutations that disrupt the FtsH3-PSST interaction . This mechanism represents a crucial component of mitochondrial protein quality control, ensuring the proper function of the respiratory chain by removing damaged Complex I components.