Recombinant Idiomarina loihiensis ATP-dependent Clp protease ATP-binding subunit ClpX (clpX)

What is Idiomarina loihiensis and what are its ecological characteristics?

Idiomarina loihiensis is a halophilic and/or haloalkaliphilic bacterium belonging to the genus Idiomarina. This organism has been isolated from diverse marine environments including hydrothermal vents and marine sediments. As a halophile, it has developed specialized adaptive mechanisms to thrive in high-salt environments. Specific strains of I. loihiensis, such as L2TR and GSL 199, have been extensively characterized through genomic analysis . The bacterium possesses unique genomic features that enable it to withstand extreme conditions, particularly high salinity and the presence of heavy metals, with adaptive mechanisms that include specialized transport systems for elements such as Fe, Cu, Zn, Pb, and Cd . Interestingly, I. loihiensis appears to preferentially metabolize proteins rather than carbohydrates, as evidenced by genomic analysis revealing incomplete carbohydrate metabolism pathways and a higher proportion of genes involved in protein metabolism .

What is the ClpX protein and how does it function in cellular processes?

ClpX is an ATP-dependent molecular chaperone belonging to the Clp/Hsp100 family of proteins. It functions primarily in two capacities: (1) as an independent chaperone that can remodel protein substrates, and (2) as a partner with ClpP, forming the ClpXP protease complex that degrades specific protein substrates . ClpX operates as a hexameric ring structure that recognizes, unfolds, and translocates proteins, using the energy from ATP hydrolysis to drive conformational changes necessary for these functions . The protein contains nucleotide-binding domains in the cleft between large and small AAA+ subdomains, which regulate the relative positioning of these subdomains during the ATP hydrolysis cycle . This conformational flexibility allows ClpX to couple ATP hydrolysis to mechanical work performed on substrate proteins. In bacterial cells, ClpX plays critical roles in protein quality control, particularly in the disassembly and degradation of aggregated proteins, as well as in the regulation of specific cellular processes such as cell division through interaction with proteins like FtsZ .

How is the ClpX protein involved in protein aggregate management?

ClpX demonstrates bona fide chaperone activity in managing protein aggregates both independently and in conjunction with ClpP. Research has shown that ClpX can target and remodel both native and non-native protein aggregates . When operating alone, ClpX can promote the disassembly and reactivation of aggregated proteins, as demonstrated with Gfp-ssrA aggregates in vitro . When partnered with ClpP to form the ClpXP complex, it facilitates both the disassembly and degradation of aggregated substrates bearing specific ClpX recognition signals . These substrates include heat-aggregated proteins like Gfp-ssrA and both polymeric and heat-aggregated forms of the bacterial cell division protein FtsZ . The ability of ClpX to discriminate between assembled and unassembled substrate conformations and to employ multivalent targeting strategies enables it to effectively remodel large oligomeric substrates. This function appears particularly important under thermal stress conditions, which promote protein aggregation. Studies in E. coli have demonstrated that cells deleted for clpX or clpP genes show exacerbated FtsZ aggregation under thermal stress, suggesting a protective role for ClpX in vivo .

How does the ATP hydrolysis mechanism of ClpX drive its chaperone and proteolytic functions?

The ATP hydrolysis mechanism of ClpX is central to its functions as both a chaperone and a component of the ClpXP protease. ClpX couples ATP binding and hydrolysis to mechanical force generation through coordinated conformational changes in its AAA+ domains . When ATP binds to ClpX protomers within the hexameric ring, it induces conformational shifts in the relative positions of the large and small AAA+ subdomains . These conformational changes are transmitted through the hexamer and converted into mechanical forces applied to substrate proteins. The ATP hydrolysis cycle proceeds through several steps:

• ATP binding to nucleotide-binding pockets in ClpX subunits

• Conformational changes that strengthen substrate engagement through the central pore

• ATP hydrolysis driving power stroke movements that pull on the engaged substrate

• ADP release and return to the initial conformation

This cyclic process enables ClpX to generate sufficient force to unfold stable protein substrates and translocate them either for remodeling (when ClpX acts alone) or into the ClpP proteolytic chamber (in the ClpXP complex). The efficiency of this mechanism is evidenced by ClpX's ability to disassemble robust protein structures such as heat-aggregated FtsZ and Gfp-ssrA . The ATP-dependent nature of these activities is clearly demonstrated in experiments where ClpX promotes the disassembly and reactivation of aggregated Gfp-ssrA through specific substrate remodeling .

What substrate recognition motifs are identified in proteins processed by I. loihiensis ClpX?

Based on studies of ClpX from other bacteria, particularly E. coli, several substrate recognition motifs are likely recognized by I. loihiensis ClpX. These recognition signals, often called degradation tags or degrons, are critical for specific substrate selection. The ssrA tag (sequence AANDENYALAA) is one of the most well-characterized ClpX recognition motifs, as evidenced by experiments using Gfp-ssrA as a model substrate . This C-terminal tag is added to incomplete proteins during translation and targets them for degradation by ClpXP.

Other recognition motifs likely include:

• C-terminal motifs: In addition to the ssrA tag, other C-terminal sequences such as the C-motif 1 (sequence VAA) and C-motif 2 (sequence LAA) may serve as recognition signals

• N-terminal motifs: Including the N-motif (sequences similar to PPWGF)

• Internal motifs: As seen in the native substrate FtsZ, which in E. coli has two distinct ClpX recognition motifs—one in the flexible linker region and one near the C-terminus

The recognition of these motifs by ClpX is the initial step in substrate processing, allowing for specific targeting of proteins for remodeling or degradation. Understanding these recognition sequences is crucial for designing recombinant proteins that can be efficiently processed by I. loihiensis ClpX in experimental systems.

What expression systems yield optimal production of functional I. loihiensis ClpX?

For optimal expression of recombinant I. loihiensis ClpX, several expression systems can be considered, with modifications to account for the halophilic nature of the source organism. While the search results don't provide specific expression protocols for I. loihiensis ClpX, established methods for similar proteins suggest the following approaches:

E. coli-based expression systems:

• BL21(DE3) strain with pET vector systems typically provides high-level expression for many recombinant proteins

• Codon optimization is advisable due to potential codon usage differences between I. loihiensis and E. coli

• Expression conditions should include:

  • Induction at lower temperatures (16-20°C) to enhance proper folding

  • Longer induction times (12-18 hours)

  • Inclusion of additional salt (0.5-1.0 M NaCl) in growth media to mimic the halophilic environment

  • Co-expression with chaperones (e.g., GroEL/ES) to assist folding

Alternative expression systems:

• Halophilic archaeal hosts (e.g., Haloferax volcanii) may provide a more native-like environment for expression

• Cell-free protein synthesis systems supplemented with high salt conditions

The selection of an appropriate expression vector should include considerations for affinity tags (e.g., His6, MBP) that facilitate purification while minimizing interference with ClpX function. Temperature, induction timing, and media composition will need empirical optimization for maximum yield of soluble, active protein.

What purification strategies yield the highest purity and activity of I. loihiensis ClpX?

A multi-step purification strategy is recommended to obtain high-purity, active I. loihiensis ClpX:

Initial Capture:

• Immobilized Metal Affinity Chromatography (IMAC) for His-tagged ClpX

  • Use buffers containing 300-500 mM NaCl to maintain protein stability

  • Include 5-10 mM ATP in buffers to stabilize the hexameric state

  • Elute with imidazole gradient (50-250 mM)

Intermediate Purification:
2. Ion Exchange Chromatography

• Anion exchange (e.g., Q-Sepharose) can exploit the likely acidic nature of this halophilic protein

• Use salt gradient elution (0.1-1.0 M NaCl)

Polishing:
3. Size Exclusion Chromatography

• Separate hexameric ClpX from monomers and aggregates

• Running buffer should contain physiological salt concentration and 1-2 mM ATP

Critical Considerations:

• Maintain 10-20% glycerol in all buffers to stabilize protein structure

• Include reducing agents (1-5 mM DTT or 2-10 mM β-mercaptoethanol) to prevent oxidation

• Add protease inhibitors during initial lysis steps

• Consider ATP concentration carefully, as it affects oligomeric state

• Monitor salt concentration throughout, as halophilic proteins may denature at low ionic strength

This purification approach has been effective for similar ATP-dependent chaperones and should yield ClpX with >95% purity while maintaining its native hexameric structure and ATPase activity.

How can researchers verify the functional activity of purified I. loihiensis ClpX?

To verify the functional activity of purified I. loihiensis ClpX, researchers should employ multiple complementary assays that assess its key activities:

ATP Hydrolysis Assays:

• Malachite green phosphate detection assay to quantify ATP hydrolysis rates

• Measure ATPase activity under varying conditions (temperature, pH, salt concentration)

• Compare activity with and without potential substrate proteins

Protein Remodeling Assays:

• Monitor the ability of ClpX to disassemble aggregated Gfp-ssrA, as demonstrated for other ClpX proteins

• Quantify reactivation of denatured luciferase or other reporter proteins

• Assess disassembly of protein polymers such as FtsZ filaments using light scattering or fluorescence microscopy techniques

ClpP Interaction Assays:

• Verify complex formation with ClpP using size exclusion chromatography or native PAGE

• Assess proteolytic activity of reconstituted ClpXP complex using fluorogenic peptide substrates

• Monitor degradation of model substrates bearing recognition tags (e.g., Gfp-ssrA)

Structural Verification:

• Confirm hexameric assembly using size exclusion chromatography, dynamic light scattering, or analytical ultracentrifugation

• Assess thermal stability using differential scanning fluorimetry at varying salt concentrations

A functional I. loihiensis ClpX should demonstrate ATP-dependent remodeling of substrate proteins, with activity parameters consistent with its adaptation to halophilic environments. Activity measurements should be performed under varying salt conditions (0.2-2.0 M NaCl) to determine optimal conditions and stability range for this halophilic enzyme.

How does I. loihiensis ClpX contribute to heavy metal resistance mechanisms?

Idiomarina loihiensis possesses remarkable heavy metal resistance capabilities, and the ClpX protein likely plays a significant role in this adaptive mechanism. The genomic analysis of Idiomarina strains reveals numerous genes associated with heavy metal tolerance, including copper resistance proteins (CopC and CopD), blue copper protein (azurin), copper binding protein (CubF), and magnesium/cobalt transport proteins (CorA and CorC) . In this context, I. loihiensis ClpX would function as a critical quality control protein that helps the bacterium cope with the proteotoxic stress caused by heavy metal exposure.

The contribution of ClpX to heavy metal resistance likely occurs through several mechanisms:

• Degradation of metal-damaged proteins: Heavy metals can cause protein misfolding and aggregation. ClpXP would selectively degrade these damaged proteins, preventing their toxic accumulation.

• Regulation of metal transport systems: ClpX may regulate the abundance of metal transporters through selective proteolysis, helping to maintain appropriate intracellular metal concentrations.

• Stress response modulation: ClpX could modulate the cellular stress response by controlling the stability of transcription factors that regulate expression of metal resistance genes.

• Maintenance of redox balance: By regulating the turnover of proteins involved in maintaining cellular redox balance, ClpX helps mitigate the oxidative stress often associated with heavy metal toxicity.

The significant role of ClpX in heavy metal resistance makes I. loihiensis ClpX a promising candidate for bioremediation applications targeting metal-contaminated environments .

What methodologies are effective for studying I. loihiensis ClpX interactions with substrate proteins?

Several complementary methodologies can effectively characterize the interactions between I. loihiensis ClpX and its substrate proteins:

In vitro interaction studies:

• Pull-down assays: Using immobilized ClpX or substrate proteins to capture interaction partners

• Surface Plasmon Resonance (SPR): For quantitative measurement of binding kinetics and affinity

• Isothermal Titration Calorimetry (ITC): To determine thermodynamic parameters of ClpX-substrate interactions

• Fluorescence Anisotropy: Using fluorescently labeled substrates to monitor binding

Substrate processing analysis:

• Gel-based degradation assays: Monitoring substrate degradation using SDS-PAGE and western blotting

• Fluorescence-based assays: Using fluorogenic substrates to monitor real-time processing

• Light scattering: To monitor disassembly of large substrate complexes and aggregates

Structural approaches:

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS): To map interaction interfaces

• Cryo-electron microscopy: To visualize ClpX-substrate complexes

• Cross-linking mass spectrometry: To identify specific contact points between ClpX and substrates

Computational methods:

• Molecular docking: To predict binding modes between ClpX and substrate recognition motifs

• Molecular dynamics simulations: To investigate the dynamics of ClpX-substrate interactions

When designing these studies, researchers should consider the halophilic nature of I. loihiensis ClpX and include appropriate salt concentrations in experimental buffers. A combination of these methodologies would provide comprehensive insights into the specificity, mechanism, and dynamics of ClpX-substrate interactions.

How can I. loihiensis ClpX be utilized in protein disaggregation studies?

I. loihiensis ClpX represents a valuable tool for protein disaggregation studies, particularly for investigations in high-salt environments. Based on studies with other ClpX proteins, I. loihiensis ClpX would be expected to promote the disassembly and potential reactivation of aggregated proteins . This capability can be leveraged in several experimental approaches:

Model system development:

• Establish assays using reporter proteins (e.g., Gfp-ssrA) that form quantifiable aggregates under stress conditions

• Create halophilic versions of model aggregation-prone proteins to study disaggregation in high-salt environments

• Develop fluorescence-based real-time monitoring systems for disaggregation kinetics

Comparative disaggregation studies:

• Compare the disaggregation efficiency of I. loihiensis ClpX with ClpX from non-halophilic organisms

• Investigate how salt concentration affects disaggregation activity

• Examine substrate specificity differences between various ClpX homologs

Mechanistic investigations:

• Analyze the ATP consumption during disaggregation to determine energetic requirements

• Study the effect of ClpX mutations on disaggregation activity to identify critical residues

• Investigate potential synergistic effects with other chaperones

Applications in biotechnology:

• Develop in vitro protein refolding systems utilizing I. loihiensis ClpX for halophilic proteins

• Design bioremediation approaches for protein-based contaminants in high-salt environments

• Create expression systems with co-expressed I. loihiensis ClpX to reduce inclusion body formation in halophilic protein production

These approaches could significantly advance our understanding of protein disaggregation mechanisms in extreme environments and provide new tools for biotechnological applications in high-salt conditions.

How do environmental factors affect I. loihiensis ClpX structure and function?

As a protein from a halophilic organism, I. loihiensis ClpX structure and function are likely significantly influenced by environmental factors, particularly salt concentration and pH. Understanding these effects is crucial for both fundamental research and biotechnological applications.

Effects of salt concentration:

pH effects:
The activity of I. loihiensis ClpX would be expected to exhibit a broader pH tolerance than non-halophilic homologs, given the haloalkaliphilic nature of some Idiomarina species . The protein likely maintains structural integrity and function across an expanded pH range of 6.0-9.0, with optimal activity potentially in the alkaline range (pH 8.0-8.5). This pH tolerance would be advantageous for applications in environments with fluctuating or extreme pH conditions.

Temperature considerations:
While specific data for I. loihiensis ClpX is not provided in the search results, proteins from marine organisms often exhibit cold adaptation. ClpX from I. loihiensis might therefore display:

• Higher specific activity at lower temperatures (10-25°C) compared to mesophilic homologs

• More flexible structure allowing catalysis at lower temperatures

• Potentially lower thermal stability at elevated temperatures (>40°C)

Metal ion effects:
Given the heavy metal tolerance of Idiomarina species , I. loihiensis ClpX likely exhibits:

• Resistance to inhibition by physiological concentrations of heavy metals

• Potential requirement for specific metal ions (e.g., Mg2+) for optimal ATPase activity

• Structural adaptations to maintain function in metal-rich environments

These environmental dependencies should be carefully considered when designing experimental protocols for studying I. loihiensis ClpX or employing it in biotechnological applications.

What challenges exist in engineering recombinant I. loihiensis ClpX for enhanced proteolytic specificity?

Engineering I. loihiensis ClpX for enhanced or altered proteolytic specificity presents several significant challenges that researchers must address:

Structural challenges:

• Limited structural information on I. loihiensis ClpX compared to model organisms

• Complex hexameric architecture with dynamic conformational states

• Difficulty in predicting how mutations affect subunit interactions and ATP hydrolysis

Substrate recognition challenges:

• Incomplete understanding of all native recognition motifs for I. loihiensis ClpX

• Multiple contact points between ClpX and substrates complicate rational design

• Potential differences in substrate preference due to halophilic adaptations

Methodological challenges:

• Maintaining protein stability during engineering processes

• Developing high-throughput screening methods compatible with high salt conditions

• Balancing enhanced specificity with catalytic efficiency

Possible engineering approaches:

• Directed evolution: Create libraries of ClpX variants and select for desired specificities

• Domain swapping: Exchange substrate-recognition loops with those from other ClpX homologs

• Rational design: Target residues in the substrate-binding pore based on molecular modeling

• Accessory domain fusion: Attach substrate-binding domains from other proteins to enhance specificity

When engineering I. loihiensis ClpX, researchers should consider developing a "toolbox" of variants with different substrate specificities, which could be valuable for various biotechnological applications, including targeted protein degradation in halophilic systems and engineered proteolytic cascades for synthetic biology applications in extreme environments.

How might genomic context and evolution inform our understanding of I. loihiensis ClpX function?

Analysis of the genomic context and evolutionary history of I. loihiensis ClpX provides valuable insights into its specialized functions and adaptations:

Genomic organization:
The clpX gene in Idiomarina species likely resides in an operon structure similar to that observed in E. coli, where clpX is located in the same operon but promoter distal to clpP . This genomic organization suggests coordinated expression of ClpX and ClpP, facilitating their assembly into the ClpXP protease complex. Analysis should include examination of:

• Regulatory elements controlling clpX expression

• Potential co-transcribed genes that may function in related pathways

• Genomic islands containing clpX, which might indicate horizontal gene transfer events

Evolutionary considerations:
Comparative genomic analysis of seven Idiomarina strains revealed 1,313 core genes related to various functions including stress response . Within this evolutionary context, ClpX likely shows:

• Sequence adaptations reflecting the halophilic lifestyle

• Conservation of key functional domains across diverse bacterial species

• Potential specialization for processing substrates unique to marine environments

Functional implications:
The evolutionary history and genomic context suggest several functional adaptations:

• Selection pressure to maintain protein quality control under salt stress

• Co-evolution with specific substrate proteins relevant to marine environments

• Adaptation to function in the presence of heavy metals commonly found in Idiomarina habitats

• Potential involvement in processing proteins related to incomplete carbohydrate metabolism pathways

Understanding these genomic and evolutionary aspects can guide research priorities and experimental designs, particularly for investigating substrate specificity, regulatory networks, and the role of ClpX in the ecological success of Idiomarina species in their unique environmental niches.

What potential roles could recombinant I. loihiensis ClpX play in bioremediation technologies?

Recombinant I. loihiensis ClpX holds significant promise for bioremediation applications, particularly for environments contaminated with heavy metals and organic pollutants. The inherent capabilities of Idiomarina species provide a strong foundation for developing ClpX-based bioremediation technologies:

Heavy metal bioremediation:
Idiomarina species possess remarkable heavy metal tolerance mechanisms, including copper resistance proteins, magnesium/cobalt transport systems, and various metal-binding proteins . Recombinant I. loihiensis ClpX could enhance these capabilities through:

• Maintaining cellular proteostasis under heavy metal stress

• Regulating the expression and turnover of metal transport and sequestration proteins

• Supporting biofilm formation, which is critical for metal biosorption and often involves curli proteins that may be processed by ClpXP

Organic pollutant degradation:
Genomic analysis of Idiomarina strains reveals genes involved in aromatic compound degradation pathways, including homogentisate and gentisate degradation pathways . ClpX could support bioremediation of these compounds by:

• Regulating the stability and activity of degradative enzymes

• Removing damaged proteins caused by exposure to toxic compounds

• Facilitating adaptation to changing pollutant concentrations

Engineered bioremediation systems:
Recombinant I. loihiensis ClpX could be integrated into advanced bioremediation technologies:

• Immobilized enzyme systems for ex situ treatment of contaminated water

• Engineered microbial consortia with enhanced degradation capabilities

• Biosensor components for monitoring environmental contaminants

The halophilic nature of I. loihiensis makes its ClpX particularly valuable for bioremediation of saline environments, such as marine oil spills, industrial effluents with high salt content, and contaminated hypersaline habitats where conventional bioremediation organisms perform poorly.

How can structural biology approaches advance our understanding of I. loihiensis ClpX?

Advanced structural biology approaches offer powerful tools to elucidate the molecular mechanisms of I. loihiensis ClpX and inform its biotechnological applications:

Cryo-electron microscopy (cryo-EM):

• Visualize the hexameric structure of I. loihiensis ClpX at near-atomic resolution

• Capture different conformational states during the ATP hydrolysis cycle

• Resolve structures of ClpX in complex with substrate proteins and ClpP

• Identify structural adaptations unique to this halophilic protein

X-ray crystallography:

• Determine high-resolution structures of individual domains

• Analyze nucleotide binding sites and potential allosteric regulation sites

• Investigate the structural basis of salt adaptation in specific protein regions

Nuclear Magnetic Resonance (NMR) spectroscopy:

• Probe dynamics of smaller domains or subunits

• Investigate conformational changes upon substrate or nucleotide binding

• Examine protein-protein interaction interfaces in solution

Integrative structural biology approaches:

• Combine multiple techniques (cryo-EM, crystallography, NMR, SAXS)

• Develop computational models validated by experimental data

• Create dynamic views of ClpX function through time-resolved structural studies

Specific research questions addressable through structural biology:

• How does the I. loihiensis ClpX hexamer assemble and maintain stability in high salt?

• What structural features enable recognition of specific substrates?

• How do conformational changes couple ATP hydrolysis to mechanical force generation?

• What structural adaptations facilitate ClpP interaction in halophilic conditions?

These structural insights would significantly advance both fundamental understanding of protein quality control in halophiles and enable rational design of ClpX variants with enhanced properties for biotechnological applications.

What future research directions might unlock novel applications for I. loihiensis ClpX?

Several promising research directions could significantly expand our understanding and application potential of I. loihiensis ClpX:

Synthetic biology integration:

• Engineer synthetic proteolytic circuits using I. loihiensis ClpX for controlled protein degradation in extreme environments

• Develop halophilic cell-free protein synthesis systems with integrated ClpX-based quality control

• Create programmable protein degradation systems for temporal control of cellular processes

Nanobiotechnology applications:

• Immobilize I. loihiensis ClpX on nanoparticles for enhanced stability and recyclability

• Develop ClpX-based nanomachines that perform mechanical work under extreme conditions

• Create biosensors using ClpX proteolytic activity as the detection mechanism

Therapeutic and pharmaceutical applications:

• Explore potential antimicrobial applications targeting essential proteins in pathogenic bacteria

• Develop enzyme replacement therapies using the salt-tolerant properties of I. loihiensis ClpX

• Investigate protein disaggregation capabilities for addressing protein misfolding diseases

Comparative systems biology:

• Compare proteostasis networks in halophiles versus mesophiles to identify unique regulatory mechanisms

• Investigate coevolution of ClpX with substrate proteins in different extreme environments

• Develop predictive models of protein quality control in halophilic bacteria

Climate change adaptation research:

• Study how I. loihiensis ClpX functions under combined stressors (high salt, temperature, pH)

• Investigate potential applications in maintaining agricultural productivity in increasingly saline soils

• Explore bioremediation applications for contaminated coastal areas affected by sea-level rise

A particularly promising direction involves leveraging the unique properties of I. loihiensis ClpX for developing biocatalysts that function in non-conventional solvents or extreme conditions, potentially enabling novel industrial bioprocesses that were previously limited by enzyme instability in harsh environments.